Combination Therapy with Gold Controlled Transgenes

ABSTRACT

Control Devices are disclosed including RNA destabilizing elements (RDE) combined with transgenes, including Chimeric Antigen Receptors (CARs) in eukaryotic cells. These RDEs can be used to optimize expression of transgenes, e.g., CARs, in the eukaryotic cells so that, for example, effector function is optimized. CARs and transgene payloads can also be engineered into eukaryotic cells so that the transgene payload is expressed and delivered at desired times from the eukaryotic cell. Such CAR T-cells with transgene payloads can be combined with the administrstion of other molecules, e.g., other therapeutics such as anticancer therapies.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CBIO046_ST25.txt”, a creation date of Jan. 24, 2020, and asize of 9 kilobytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Chimeric Antigen Receptors are human engineered receptors that maydirect a T-cell to attack a target recognized by the CAR. For example,CAR T cell therapy has been shown to be effective at inducing completeresponses against acute lymphoblastic leukemia and other B-cell-relatedmalignancies and has been shown to be effective at achieving andsustaining remissions for refractory/relapsed acute lymphoblasticleukemia (Maude et al., NEJM, 371:1507, 2014). However, dangerous sideeffects related to cytokine release syndrome (CRS), tumor lysis syndrome(TLS), B-cell aplasia and on-tumor, off-target toxicities have been seenin some patients.

There are currently two extant strategies to control CAR technology. Thefirst is an inducible “kill switch.” In this approach, one or more“suicide” genes that initiate apoptotic pathways are incorporated intothe CAR construct (Budde et al. PLoS1, 2013doi:10.1371/journal.pone.0082742). Activation of these suicide genes isinitiated by the addition of AP1903 (also known as rimiducid), alipid-permeable tachrolimus analog that initiates homodimerization ofthe human protein FKBP12 (Fv) to which the apoptosis-inducing proteinsare translationally fused In the ideal scenario, these kill switchesendeavor to sacrifice the long-term surveillance benefit of CARtechnology to safeguard against toxicity. However, in vivo, thesesuicide switches are not likely to realize this goal, as they areoperating against powerful selection pressures for CAR T-cells that donot respond to AP1903, a situation worsened by the inimical error-proneretroviral copying associated with the insertion of stable transgenesinto patient T-cells. In this scenario, non-responsive CAR T-cell cloneswill continue to proliferate and kill target cells in anantigen-dependent manner. Thus, kill switch technology is unlikely toprovide an adequate safeguard against toxicity.

The second CAR regulatory approach is transient CAR expression, whichcan be achieved in several ways. In one approach, T-cells are harvestedfrom unrelated donors, the HLA genes are deleted by genome-editingtechnology and CAR-encoding transgenes are inserted into the genome ofthese cells. Upon adoptive transfer, these CAR T-cells will berecognized by the recipient's immune system as being foreign anddestroyed, thus the CAR exposure in this system is transient. In anothertransient CAR exposure approach, mRNA of a CAR-encoding gene isintroduced into harvested patient T-cells (Beatty, G L 2014. CancerImmunology Research 2 (2): 112-20. doi:10.1158/2326-6066.CIR-13-0170).As mRNA has a short half-life and is not replicated in the cell orstably maintained, there is no permanent alteration of theCAR-expressing T-cell, thus the CAR expression and activity will be fora short period of time. However, as with the kill-switch approach, thesetransient CAR exposure approaches sacrifice the surveillance benefit ofCARs. Additionally, with these transient systems acute toxicity can bedifficult to control.

SUMMARY OF THE INVENTION

In an aspect, the description discloses new chimeric antigen receptors(CARs) that use a knottin (an inhibitor cystine-knot protein or cysteineknot protein) as the antigen recognition portion of the CAR. Knottinscan be engineered to recognize targets of interest as knottins have acore structure (scaffold) with peptide loops around the core structurewhich peptide loops can be engineered to produce different bindingproperties and specificities. Knottins can be small and so have theproperties of a small molecule with the binding affinity of a biologic(such as an antibody). Knottins have high stability to pH, heat, andenzymes.

In an aspect, the description discloses a eukaryotic cell with a CAR,T-cell receptor, or other targeting polypeptide and a transgene underthe control of an RNA Destabilizing Element (RDE). The RDE may controlmultiple transgenes or multiple RDEs may control multiple transgenes.The multiple transgenes may be arranged serially and/or as a concatemerand/or in other arrangements. Multiple RDEs may be used to regulate atransgene, and these multiple RDEs can be organized as a concatemer,interspersed within a region of the transcript, or located in differentparts of the transcript. Multiple transgenes can be regulated by an RDEor a combination of RDEs. The RDEs can be localized in the 3′-UTR, the5′-UTR and/or an intron. RDEs can include, for example, the RDEs from AU1 (CD40L), AU 2 (CSF2), AU 3 (CD247), AU 4 (CTLA4), AU 5 (EDN1), AU 6(IL2RA), AU 7 (SLC2A1), AU 8 (TRAC), AU 9 (CD274), AU 10 (Myc), AU 11(CD19), AU 12 (IL4), AU 13 (IL5), AU 14 (IL6), AU 15 (IL9), AU 16(IL10), AU 17 (IL13), AU 18 (FOXP3), AU 19 (TMEM-219), AU 20(TMEM-219snp), AU 21 (CCR7), AU 22 (SEM-A4D), AU 23 (CDC42-SE2), AU 24(CD8), AU 27 (bGH), and/or AU 101 (Interferon gamma or IFNg). Other RDEsare disclosed in the following description. RDE control can also becombined with codon optimization of the transgene to increase the GCcontent of the wobble position (third position of the codon) in some orall of the codons of the transgene. This codon optimization can increaseefficiency of expression (the on signal) by up to 100-fold. Such codonoptimized transgenes can be linked to an RDE and produce a largerdynamic range of expression from the RDE control compared to thetransgene-RDE without codon optimization.

In an aspect, the RDE can be under the control of a RNA control device.Such, Smart RDEs place the RDE control under the regulation of the RNAcontrol device which introduces ligand control to the RDE. The RNAcontrol device can disrupt the RDE when ligand is bound (or not bound)resulting in loss of the RDE control, and when ligand is added (orremoved) the RNA control device is inhibited and the RDE structure isavailable for interaction with RNA binding proteins. The RNA controldevice could also act upon a portion of the transcript that disrupts theRDE (e.g., the portion of the transcript could form secondary structureswith the RDE that inhibit RNA binding proteins from binding to the RDE),when the RNA control device binds (or is free from ligand) the RNAcontrol device disrupts the inhibitory portion of the transcript so itis not available to interact with the RDE, and the RDE is now availableto interact with RNA binding proteins. This RNA control deviceregulation allows the activity of the RDE to be ligand controlledthrough the action of the RNA control device.

In an aspect, a therapy utilizing a CAR T-lymphocyte with or without anRDE controlled transgene(s) is combined or in an order of successionwith another therapy. The other therapy can include any therapeuticmolecule including, for example, a polypeptide, lipid, carbohydrate,nucleic acid, small molecule drug, biological drug, antibody,antibody-drug-conjugate, or combinations of the foregoing. Suitablemolecules are described below. The other therapy can be administered toa subject at the same time as the CAR therapy (with or without a RDEcontrolled transgene(s)), before the administration of the CAR therapy(with or without a RDE controlled transgene(s)), or after theadministration of the CAR therapy (with or without a RDA controlledtransgene(s)). For example, a subject could be treated with chemotherapyand/or an immunotherapy (e.g., an antibody-drug conjugate), followed bytreatment with a CAR T-cell with optionally a RDE controlled payload.The CAR T-cell treatment can be given the subject at varying times afterthe chemotherapy and/or immunotherapy, e.g., one, two, three, four,five, or six weeks. The chemotherapy and/or immunotherapy (e.g., ADC)can be cycled with the CAR T-cell treatment for multiple cycles oftreatment. Treatment with CAR T-cells may also be boosted with target Xpeptide, or virus or cells loaded with target X peptide (target X is thetarget bound by the CAR).

In an aspect, an RDE, combination of RDEs, and/or modified RDEs can beused to provide desired kinetic parameters to the regulation of a geneproduct including, for example, amount of expression, steady stateconcentration, C_(max) (maximal concentration of gene product obtained),T_(max) (time to reach C_(max)), baseline expression, speed of induction(acceleration), induction rate (velocity), dynamic range also known asfold regulation (induced expression/basal expression), maximal dynamicrange (DR_(max)), time to DR_(max), area under the curve (AUC), etc. ARDE construct can be made that has a desired set of kinetic parametersto provide the level, degree, temporal, and amount of regulation that isdesired. In addition, RDE concatemers can be used to alter the kineticperformance of a construct.

Combinations of RDEs can be used to provide temporal regulation betweentwo or more transgenes. RDEs can be selected to provide maximal rates ofexpression (and different amounts of maximal expression) at differenttimes following activation of a cell (or induction of expression). Thistemporal control allows a first transgene encoded polypeptide to alterthe state of the cell so that the cell is prepared to be acted upon by asecond polypeptide encoded by a second transgene with an RDE thatprovides later in time expression. This temporal control can also beused to time the expression of two, three or more transgenes followingactivation of a cell. If the transgene encoded polypeptides aresecreted, they can act in a temporal fashion upon target cells. Forexample, a first transgene polypeptide (with an early expression RDE)could be secreted and act upon a target cell to change its state (e.g.,induce the expression of receptor). The second transgene polypeptide isexpressed at a later time (under the control of a later expression RDE)and acts upon the target cell with the changed state (e.g., the secondprotein can be a ligand for the induced receptor).

In an aspect, the RDE can be engineered to increase or decrease thebinding affinity of RNA binding protein(s) that interact with the RDE.Altering the affinity of the RNA binding protein can change the timingand response of transgene expression as regulated by the RNA bindingprotein. In an aspect, the RNA binding protein binding at the RDE isaltered by the metabolic state of the cell and changing the bindingaffinity of the RDE for the RNA binding protein alters the response toand/or timing of transgene expression with the metabolic state of thecell. In an aspect, the RNA binding protein binding at the RDE isaltered by the redox state of the cell and changing the binding affinityof the RDE for the RNA binding protein alters the response to and/ortiming of transgene expression with the redox state of the cell.

In an aspect, the CAR, T-cell receptor, B-cell receptor, innate immunityreceptor, or other targeting receptor or targeting polypeptiderecognizes an antigen at the target site (e.g., tumor cell or otherdiseased tissue/cell) and this activates the cell. The transgene can beanother CAR that recognizes a second antigen at the target site andactivation of the cell by the first CAR, T-cell receptor or othertargeting polypeptide induces the second CAR allowing the eukaryoticcell to recognize the target site by a second antigen. In an aspect, theeukaryotic cell has a first CAR that recognizes an antigen at a targetsite and this activates a transgene (through an RDE) that encodes apolypeptide that directly or indirectly reduces the activation state ofthe cell. For example, the transgene may encode a second CAR thatrecognizes an antigen on healthy tissue so that when the first CARreacts with antigen at a nontarget cell, the eukaryotic cell will bede-activated by the second CAR interaction with the healthy cell antigen(that is not present or is present in reduced amounts at the targetsite).

In some aspects, the eukaryotic cell is an immune cell, e.g., a T-cell,a natural killer cell, a B-cell, a macrophage, a dendritic cell, orother antigen presenting cell. In these aspects, activation of the cellby the CAR or changing the metabolic state of the immune cell in otherways can induce expression of the transgene through the RDE. The RDEthat controls the transgene can have microRNA binding sites and can beengineered to remove one or more of these microRNA binding sites. TheRDE can be bound by the Hu Protein R (HuR). Without wishing to be boundby theory it is expected that HuR can bind to some RDEs, and act tostabilize the mRNA, leading to enhanced translation. Some RDEs can betied to the glycolytic state of the eukaryotic cell through the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH), other dehydrogenases,other oxidoreductases, or other glycolytic enzymes that can bind to anRDE when the eukaryotic cell is not activated (low glycolytic activity),quiescent, or at rest. When GAPDH or the other enzymes bind to the RDEthis can reduce half-life of the RNA with the RDE. In this aspect, CARactivation of the eukaryotic cell (e.g., T-lymphocyte) can induceglycolysis in the cell which reduces GAPDH binding of the RNA, increaseshalf-life of the RNA, which produces increased expression of thetransgene encoded in the RNA and controlled by the RDE. Without wishingto be bound by theory, as GAPDH vacates the RDE, HuR or other RDEbinding proteins may subsequently bind either the same RDE, or apreviously inaccessible RDE (sterically hindered by presence of GAPDH),further stabilizing the mRNA, increasing half-life of the mRNA, andproducing further increased expression of the transgene encoded by theRNA and controlled by said RDE. Thus, CAR activation can induceexpression of the transgene. In other aspects, other activation of theimmune cell can cause GAPDH to engage in glycolysis and so induceexpression of the transgene under the control of the RDE. Examples ofRDEs bound by GAPDH include, for example, AU 19 (TMEM-219), AU 20(TMEM-219snp), AU 21 (CCR7), AU 22 (SEM-A4D), and AU 23 (CDC42-SE2).

Expression from the transcript with the RDE(s) can respond to themetabolic state of the cell. For example, the RDE can be bound bymetabolic or glycolytic enzymes which couples expression of thetransgene to the activation state of the cell through these metabolic orglycolytic enzymes. Some metabolic or glycolytic enzymes bind to RDEs inthe transcript and degrade or target for degradation the transcript.When those metabolic or glycolytic enzymes become active, the enzymes nolonger bind to the RDEs, the transcripts are stable for a longer periodof time, and the transcripts can be translated for this longer period oftime. Cells expressing transgenes under the control of such RDEs canalso be engineered to express a CAR that can alter the metabolic stateof the cell at desired times resulting in expression of the transgene atthe desired time. Alternatively, other stimuli can be used to alter themetabolic state of the eukaryotic cell resulting in expression of thetransgene. For example, the metabolic state of the cell can be alteredto cause transgene expression (or to inhibit expression) by stimuliincluding, for example, small molecules (e.g., PMA/ionomycin),cytokines, a TCR and costimulatory domain engagement with ligand, oxygenlevels, cellular stress, temperature, or light/radiation.

GAPDH binding to the RDE can be increased by introducing into the cell asmall molecule that inhibits glycolysis such as, for example,dimethylfumarate (DMF), rapamycin, 2-deoxyglucose, 3-bromophyruvic acid,iodoacetate, fluoride, oxamate, ploglitazone, dichloroacetic acid, orother metabolism inhibitors such as, for example,dehydroepiandrosterone. Other small molecules can be used to reduceGAPDH binding to the RDE. Such small molecules may block the RDE bindingsite of GAPDH including, for example, CGP 3466B maleate or Heptelidicacid (both sold by Santa Cruz Biotechnology, Inc.), pentalenolactone, or3-bromopyruvic acid. Other small molecules can be used to analogouslyinhibit other enzymes or polypeptides from binding to RDEs. Other smallmolecules can be used to change the redox state of GAPDH, leading to analtered affinity of GAPDH for the RDE. Other small molecules known tointeract with GAPDH function, such as vitamin C, saframycin, salicylicacid, insulin, vitamin d3, metformin, or suramin can modify the bindingof GAPDH for the RDE. Other molecules can modify the binding of GAPDHfor the RDE including trehalose, galactose and other saccharides. Othermolecules known to alter GAPDH structure can modify the binding of GAPDHfor the RDE including nitric oxide and hydrogen sulfide.

GAPDH binding of RDEs can also be modified by acetyl-CoA. GAPDH can beacetylated at a number of Lysines in the GAPDH sequence. Bond et al,FASEB J. 31:2592-2602 (2017), which is incorporated by reference in itsentirety for all purposes. Acetylation at Lysine 254 can increase GAPDHactivity in response to glucose. ATP citrate lyase (ACLY) can increasethe level of acetyl-CoA in a cell thereby increasing the acetylation ofGAPDH. ACLY can be expressed in eukaryotic cells (e.g., an immune cellsuch as a T-cell) at a desired time which can increase acetylation ofGAPDH which can increase expression from RDE controlled transcripts. Forexample, ACLY can be used as a payload regulated by an RDE. In thisexample, a eukaryotic cell (e.g., an immune cell) is activated and theRDE controlled ACLY is expressed. The ACLY can increased acetylation ofGAPDH which can cause more expression from RDE controlled transcripts.In this example, if another payload is under control of an RDE theincrease in expression that payload can be increased by theco-expression of ACLY as acetylation reduces the GAPDH bound to RDEs.

In an aspect, alternative splicing is used to complement or in lieu ofRDE control to tie cell activation and/or change in metabolic state tothe expression of a transgene. For example alternative splicing mediatedby hnRNPLL can be used. hnRNPLL is expressed when immune cells (e.g.,T-cells or B-cells) are activated. hnRNPLL binds to RNA transcripts andcan cause the retention or excision of introns and/or exons resulting inalternative splicing of RNAs (e.g., mRNAs). hnRNPLL alternative splicingcontrol can be used alone or in combination with RDE control to regulatethe expression of transgenes. When a cell is activated (e.g., by ligandbinding at a receptor which can change a cell's metabolic state) it caninduce expression of hnRNPLL, which can produce alternative splicingproducts in transcripts with hnRNPLL binding sites. As discussed aboveand below, such cell activation also induces expression in transcriptswith RDE sites. By combining RDE control with hnRNPLL control thedynamic range of control for a transgene upon cell activation (e.g.,receptor binding of ligand) can be increased.

In an aspect, activation of the immune cell induces expression of thetransgene that can encode a payload to be delivered at the target(activation) site. The transgene can encode a payload for delivery atthe site of CAR activation and/or immune cell activation and/or otherreceptor activation. The payload can be a cytokine, an antibody, areporter (e.g., for imaging), a receptor (such as a CAR), or otherpolypeptide that can have a desired effect at the target site. Thepayload can remain in the cell, or on the cell surface to modify thebehavior of the cell. The payload can be an intracellular protein suchas a kinase, phosphatase, metabolic enzyme, an epigenetic modifyingenzyme, a gene editing enzyme, etc. The payload can be a gene regulatoryRNA, such as, for example, siRNA, microRNAs (e.g., miR155), shRNA,antisense RNA, ribozymes, and the like, or guide RNAs for use withCRISPR systems. The payload can be a nucleic acid (e.g., a vector, or ahuman artificial chromosome (HAC)). The payload can also be a membranebound protein such as GPCR, a transporter, etc. The payload can be animaging agent that allows a target site to be imaged (target site has adesired amount of target antigen bound by the CAR). The payload can be acheckpoint inhibitor, and the CAR and/or other binding protein (e.g.,T-cell receptor, antibody or innate immunity receptor) can recognize atumor associated antigen so the eukaryotic cell preferentially deliversthe checkpoint inhibitor at a tumor. The payload can be a cytotoxiccompound including, for example, a granzyme, an apoptosis inducer, acytotoxic small molecule, or complement. The payload can be an antibody,such as for example, an anti-4-1BB agonist antibody (an anti-CD137antibody), an anti-IL 1b antibody (anti-inflammatory),anti-CD29/anti-VEGF antibody, an anti-CTLA4 antibody, a bispecificantibody (e.g., BiTE), or an anti-CD11b antibody. The payload can be animmune polypeptide, including for example, cytokines (e.g., IL-2, IL-12,IL-15, IL-18), chemokines (e.g., CXCL12), perforins, granzymes, andother immune polypeptides. The payload can be an enzyme including forexample, hyaluronidase, or heparinase. The payload can be a polypeptideincluding for example, ApoE (e.g., ApoE2, ApoE3 and ApoE4), NO synthase(e.g., iNOS, nNOS, eNOS), HSV-thymidine kinase (HSV-TK), antagonists ofCSF1 receptor, CCR2, CCR4, a BiTE (activates immunosuppressed T-cells),soluble CD40 ligand, HSP70, and HSP60. The payload can be fused orassociated with Decorin, Biglycan, fibromodulaon/Lumican so that thepayload binds to the collagen near or in the target site. This strategyis particularly useful for keeping cytotoxic payloads localized to thetarget cells (e.g., a tumor). The payload can be a transgene(s) whichdelivers a virus as a payload. For example, the RDE can control a mastercontrol element that controls the expression of the virus genes forreplication and coat/envelope proteins. Alternatively, the Rep andcoat/envelope proteins can be placed under the control of induciblepromoters that are controlled by a regulatory protein, and thatregulatory protein can be controlled by an RDE. Still alternatively, theRep proteins of the virus can be placed under the control of an RDE,and/or the coat/envelope proteins of the virus can be placed under thecontrol of an RDE. As with other payloads this complex payload can useCAR T-cell regulation or any other regulation that induces glycolysis ina cell. Helper constructs in a T cell, or other delivery cell can encodethe genes needed for viral replication and viral packaging.

In some aspects, the expression of CAR, DE-CAR and/or Side-CARpolypeptide is controlled, at least in part, by an RDE that interactswith a glycolytic enzyme with RDE binding activity, e.g., GAPDH. Theglycolytic enzyme can bind to the RDE and reduce production of the CAR,DE-CAR, Side-CAR polypeptide, and/or other transgene product. Thisreduction in polypeptide production can occur because of an inhibitionof translation and/or an increase in the rate of mRNA degradation (RDEbinding can shorten the half-life of the mRNA). Some RDE bindingproteins may reduce translation and enhance degradation of RNA to reducethe level of polypeptide made. The RDE can be an AU rich element fromthe 3′ UTR of a transcript (e.g., a transcript encoding IL-2 or IFN-γ),or can be a modified 3′ UTR that has been engineered to remove one ormore microRNA sites (e.g., modified 3′-UTRs of IL-2 or IFN-γ). In anaspect, the expression of the transgene, CAR, DE-CAR and/or Side-CARpolypeptide under the control of an RDE bound by a glycolytic enzyme(s),e.g., GAPDH, is increased by increasing the activity of the enzyme(s) inprosecuting glycolysis. The activity of enzymes in glycolysis can beincreased by providing the cell with increased glucose in the cellmedium, increasing triose isomerase activity in the cell, or providingthe cell with a compound that increases glycolysis in the cell, e.g.,tamoxifen or glucose. The RDE can bind to Hu Protein R (HuR). Withoutwishing to be bound by theory it is expected that HuR binds to someAU-rich RDEs and U-rich RDEs, and can act to stabilize the mRNA, leadingto enhanced translation. Thus, cell conditions that result in increasedHuR expression can increase expression of transgenes with appropriateAU-rich elements and/or U-rich elements, and conditions that reduce HuRexpression can decrease expression of these transgenes. HuR interactionwith the 3′ UTR of the transgene (or native genes) can also be alteredby expressing a recombinant transcript containing HuR binding sites.Expression of these transcripts will reduce the amount of HuR availableto bind to the transgene transcript or native HuR regulated transcriptsand reduce the half-lives of these transcripts resulting in decreasedexpression.

In an aspect, RDE control can be used to lower CAR expression in asubject which can reduce the availability of CAR polypeptide for immunereactions. This can lower the immunogenicity of transgenic immune cellswith the CAR. In part, this lower immunogenicity occurs because the CARpeptide has lower exposure to the immune system.

In an aspect, nucleic acids can be used to boost the response of immunecells upon stimulation of the immune cell. For example, the immune cellcan produce higher amounts of immune polypeptides (greater C_(max)) withfaster kinetics of production. The immune polypeptides can include, forexample, cytokines, perforins, granzymes, apoptosis inducingpolypeptides, etc. The nucleic acids that boost the immune response cancomprise control regions operably linked to nucleic acids encoding RDEsfor selected RDE binding proteins, so that upon expression of thenucleic acid into RNA the RDEs in the RNA bind the RDE binding proteinsthat repress expression of a polypeptide, for example, cytokines,perforins, granzymes, and other immune polypeptides. The expression ofthe RNAs with the RDEs can poise the eukaryotic cell for expression ofpolypeptide controlled by RDEs. For example, the expression of RNAs withthe RDEs may be done in immune cells to poise the cell for expression ofimmune polypeptides upon stimulation of the immune cell.

In an aspect, the CAR, DE-CAR, Side-CAR polypeptides, and/or otherreceptor can be directed against antigens found on acute myeloidleukemia (AML) cells including, for example, CD 33, CD 34, CD 38, CD43,CD 44, CD 45, CD 45RA, CD 47, CD 64, CD 66, CD 123, CD 133, CD 157,CLL-1, CXCR4, LeY, PR1, RHAMM (CD 168), TIM-3, and/or WT1. Themonoclonal antibody 293C3-SDIE can be used as the extracellular elementfor the CAR, DE-CAR and/or Side-CAR polypeptides. (Rothfelder et al.,2015, at ash.confex.com/ash/2015/webprogram/Paper81121.htmh which isincorporated by reference in its entirety for all purposes) Otherantigens for AML are known in the art and may be the target of the CAR,DE-CAR, Side-CAR, and/or other receptor. An onco-sialylated CD 43 hasbeen associated with acute myeloid leukemia (AML) and thisonco-sialylated CD 43 is not found on normal cells and tissue. Thisonco-sialylated CD 43 is bound by the monoclonal antibody AT14-013, andthe variable region of this antibody is used to make an anti-oncosialylated CD 43 CAR. AT14-013 recognizes the unique sialylation epitopefound on this onco-sialylated CD 43. This CAR is specific for AML anddoes not have side reactivity with normal tissue in a subject. In anaspect, the CAR, DE-CAR, Side-CAR polypeptides, and/or other receptorcan be directed against antigens found on diffuse large cell B-celllymphoma (DLBCL) cells including, for example, CD19, CD20, CD22, CD79a,CD5, CD10, and CD43. Other antigens for DLBCL are known in the art andmay be the target of the CAR, DE-CAR, Side-CAR, and/or other receptor.

Other antigens that can be targeted by the CAR, DE-CAR, side-CAR orother receptor include, for example, DLL3, HER2, PSCA, CSPG4, EGFRvIII,MSLN (mesothelin), FAP, MUC16 (CA-125), CEA, CD133 (PROM1), IL13Ra,CD171 (L1CAM), CD123, (IL3R), CD33 (SIGLEC3), LeY, GUCY2C, BCMA and/orEPHA2. Eukaryotic cells with CAR, DE-CAR, side-CAR or other receptorstargeting these antigens can include a payload such as, for example, oneor more of anti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4antibody, anti-IL1b antibody, a BiTE, CCL2, anti-CXCR4 antibody,anti-CXCL12 antibody, HAC, heparinase, hyaluronidase, Hsp60, Hsp70,IL-2, IL-12, IL-15, IL-18, INFγ, miRNA (e.g., mir155), CD40 ligand,ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12, ACLY, antagonists of CSF1receptor, siRNA, other antisense RNAs, and/or anti-CD28 antibody(including full length and fragments such as single chain antibodies).The payload can be presented as a fusion protein with a Small LeucineRich Proteoglycans (SLRPs) such as Decorin, Biglycan and/or Lumican (allof which can anchor the fusion protein to the cellular matrix in or nearthe target site). Fusions with Decorin and/or Biglycan can also bindTGF-beta in and around the target site which can reduce immunesuppression. Myeloid modifying payloads (“MM payloads”) which reduceimmune suppression or inhibition caused by myeloid cells may bedelivered including, for example, ApoE3, ApoE4, Hsp60, Hsp70, TNFα,antagonists of CSF1 receptor, CD40L (CD154) and/or IL-12. Two or more MMpayloads can also be delivered by the CAR, DE-CAR, side-CAR and/or otherreceptor cell (e.g., T-cell) using RDEs that produce differentpharmacokinetics for delivery. For example, the different MM payloadscould be controlled by different RDEs so that the Cmax of delivery forthe different MM payloads occurs at different times.

AB toxins can be used to engineer fusion payloads that deliver a desiredprotein product into a target cell. AB toxins include, for examplediphtheria toxin, tetanus toxin, exotoxin A of P. aeruginosa, iota toxinIa of C. perfringens, C2 toxin CI of C. botulinum,ADP-ribosyltransferase of C. difficile, etc. The AB toxin can beengineered to replace the catalytic (toxic) component (A domain) of theAB toxin with the desired protein so that the modified AB toxin bindsits receptor and delivers the desired protein into a target cell throughthe B domain of the AB toxin. These B toxin fusions can also beengineered to replace the receptor binding portion of the B domain witha binding domain (e.g., ligand) that binds to a desired antigen and/orreceptor on a desired target cell. These modified B toxin fusions candeliver a desired protein payload into a target cell using the B domainof the toxin.

The CAR, DE-CAR, Side-CAR polypeptides, and/or other receptor can bedirected against antigens found on solid tumors such as, for example,integrins such as αvβ6 (found on numerous solid tumors (including, forexample, oral squamous cell cancer, colon cancer, pancreatic cancer,gastric cancer, breast cancer, ovarian cancer, cervical cancer, lungcancer, etc.).

In an aspect, small molecules and other molecules that affect theavailability of GAPDH or other RDE binding proteins to bind RDEs can beused to regulate gene expression by GAPDH, other RDE binding glycolysisenzymes, and/or other RDE binding enzymes involved in energy and cellmetabolism. Molecules that increase glycolysis in a cell can reduce theamount of GAPDH available for binding to RDEs which can increasetranslation from transcripts under GAPDH control. Similarly, otherglycolysis enzymes and metabolic enzymes can bind to RDEs and activatingglycolysis and other energy pathways in the cell can reduce the amountof these enzymes that are available to bind their corresponding RDEs.This reduced binding can increase translation from transcriptscontrolled by these RDE binding proteins (enzyme binding to the RDEdecreases expression) or can decrease translation if enzyme binding hasa positive effect on expression. These molecules can also be useful inthe treatment of certain types of neural degeneration associated withinflammation and/or autoimmune diseases. These molecules can be used toalter the amount of GAPDH in immune cells so that RDEs are bound and theimmune cells reduce expression of RDE regulated genes. Some genes underRDE control in immune cells are associated with inflammation and so,molecules that increase the amount of RDE binding proteins that inhibitthe inflammatory associated transcripts could reduce inflammation.

A nucleic acid construct encoding a transcript with selected RDEs can beexpressed in an immune cell, for example, a T-lymphocyte. Therecombinant transcript with the selected RDEs can bind to and depletethe levels of RDE binding proteins in the T-lymphocyte so thattranscripts encoding polypeptides regulated by the depleted RDE bindingproteins are expressed at different threshold points of activation forother cellular signals. The use of the RDE constructs can increase thekinetics of expression and/or the Cmax of expression of the polypeptideswhose expression is controlled by the RDE.

In an aspect, CAR T-lymphocytes are modified to reduce or prevent graftversus host reactions. This is done by knocking out the ability of theT-cell receptors to activate the T-lymphocyte. When a dominant CD3epsilon chain mutant is introduced to the T-lymphocyte it can knockoutthe ability of the T-cell receptors to activate the T-lymphocyte. Onesuch CD3 epsilon mutant is a double mutant that replaces the Cysteineresidues of the C-X-X-C motif with Serine residues (a C119S and C122Smutant). This mutant CD3 epsilon chain disrupts signaling from theT-cell receptor and prevents ligand induced activation of theT-lymphocyte through the T-cell receptor. By expressing an excess of theCD3 epsilon C119S/C122S mutant in a T-lymphocyte, the T-lymphocyte willnot activate in response to host antigens binding to the T-lymphocytereceptor of the CAR T-lymphocytes. This allows the use of allogenic CART-lymphocytes in a subject with reduced graft versus host reactions.

In an aspect, an expression vector has a CAR cassette and a CD3 epsilonmutant (a dominant mutant such as C119S/C122S) cassette. These twoexpression cassettes are operably linked to a strong promoter (e.g.,with a strong enhancer active in T-lymphocytes). The two cassettes canhave different promoters or can be expressed from the same promoter. Theexpression construct can also include a transgene under control of adifferent promoter, which transgene is operably linked to a RDE thatactivates transgene expression upon stimulation of the cell by the CAR.

In an aspect, Tox expression in a T-lymphocyte or T-cell is regulated toproduce either an effector state (low Tox) or an exhausted state (T_(ex)with high Tox). Tox can be down regulated in activated T-lymphocyteswith a miRNA, siRNA or antisense RNA targeted at Tox. The Tox locus inan engineered T-lymphocyte can also be knocked out so that it does notproduce a functional product. Conversely, Tox can be expressed in anengineered T-lymphocyte to exhaust the cell and turn it off (an offswitch).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram for optimal CAR activity where the threevariables are CAR copy number, target epitope copy number and CARbinding affinity.

FIG. 2 shows a graph for the bioluminescence from T-cells withluciferase controlled by an RDE following activation of the T-cell byRaji target cells (activate CAR) or by CD3/CD28 beads (activate TCR) ascompared to bioluminescence of T-cells at resting.

FIG. 3 shows a graph for bioluminescence from T-cells with luciferasecontrolled by the RDEs Gold1, Gold2, or Gold3 following activation ofthe T-cell by Raji target cells (activate CAR) as compared tobioluminescence of T-cells at resting.

FIG. 4 shows a graph for the IL-12 expression from T-cells with IL-12expression controlled by an RDE following activation of the T-cell byRaji target cells (activate CAR) as compared to IL-12 expression ofT-cells at resting.

FIG. 5 shows basal luciferase expression and activated luciferaseexpression for luciferase constructs utilizing different RDEs as controlelements in Jurkat cells.

FIG. 6 shows basal luciferase expression and activated luciferaseexpression for luciferase constructs utilizing different RDEs as controlelements in primary T-cells.

FIG. 7 shows activated luciferase/basal luciferase expression after 1,3, 6, and 8 days for luciferase constructs utilizing different RDEs ascontrol elements.

FIG. 8 shows basal luciferase expression and activated luciferaseexpression for luciferase constructs utilizing different RDEs as controlelements.

FIG. 9 shows the dynamic range (activated luciferase/basal luciferase)measured 1, 3/4, 6, and 8 days after activation for luciferaseconstructs utilizing different RDEs as control elements.

FIG. 10 shows the dynamic range (activated luciferase/basal luciferase)measured 1, 3/4, 6, and 8 days after activation for luciferaseconstructs utilizing different RDEs as control elements.

FIG. 11 shows the impact on luciferase expression for luciferaseconstructs utilizing an RDE as a control element in the presence ofglucose and galactose.

DETAILED DESCRIPTION OF THE INVENTION

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimscan be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.Numerical limitations given with respect to concentrations or levels ofa substance are intended to be approximate, unless the context clearlydictates otherwise. Thus, where a concentration is indicated to be (forexample) 10 μg, it is intended that the concentration be understood tobe at least approximately or about 10 μg.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, an “actuator element” is defined to be a domain thatencodes the system control function of the RNA control device. Theactuator domain can optionally encode the gene-regulatory function.

As used herein, an “antibody” is defined to be a protein functionallydefined as a ligand-binding protein and structurally defined ascomprising an amino acid sequence that is recognized by one of skill asbeing derived from the variable region of an immunoglobulin. An antibodycan consist of one or more polypeptides substantially encoded byimmunoglobulin genes, fragments of immunoglobulin genes, hybridimmunoglobulin genes (made by combining the genetic information fromdifferent animals), or synthetic immunoglobulin genes. The recognized,native, immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes and multiple D-segments andJ-segments. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. Antibodies exist as intact immunoglobulins, as a number ofwell characterized fragments produced by digestion with variouspeptidases, or as a variety of fragments made by recombinant DNAtechnology. Antibodies can derive from many different species (e.g.,rabbit, sheep, camel, human, or rodent, such as mouse or rat), or can besynthetic. Antibodies can be chimeric, humanized, or humaneered.Antibodies can be monoclonal or polyclonal, multiple or single chained,fragments or intact immunoglobulins.

As used herein, an “antibody fragment” is defined to be at least oneportion of an intact antibody, or recombinant variants thereof, andrefers to the antigen binding domain, e.g., an antigenic determiningvariable region of an intact antibody, that is sufficient to conferrecognition and specific binding of the antibody fragment to a target,such as an antigen. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, and Fv fragments, scFv antibodyfragments, linear antibodies, single domain antibodies such as sdAb(either VL or VH), camelid VHH domains, and multi-specific antibodiesformed from antibody fragments. The term “scFv” is defined to be afusion protein comprising at least one antibody fragment comprising avariable region of a light chain and at least one antibody fragmentcomprising a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the VL and VH variable regions in either order,e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise VL-linker-VH or may compriseVH-linker-VL.

As used herein, an “antigen” is defined to be a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including, but not limited to, virtually all proteins orpeptides, including glycosylated polypeptides, phosphorylatedpolypeptides, and other post-translation modified polypeptides includingpolypeptides modified with lipids, can serve as an antigen. Furthermore,antigens can be derived from recombinant or genomic DNA. A skilledartisan will understand that any DNA, which comprises a nucleotidesequences or a partial nucleotide sequence encoding a protein thatelicits an immune response therefore encodes an “antigen” as that termis used herein. Furthermore, one skilled in the art will understand thatan antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be synthesized or can be derived from a biologicalsample, or can be a macromolecule besides a polypeptide. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a fluid with other biological components.

As used herein, the terms “Chimeric Antigen Receptor” and the term “CAR”are used interchangeably. As used herein, a “CAR” is defined to be afusion protein comprising antigen recognition moieties andcell-activation elements.

As used herein, a “CAR T-cell” or “CAR T-lymphocyte” are usedinterchangeably, and are defined to be a T-cell containing thecapability of producing CAR polypeptide, regardless of actual expressionlevel. For example a cell that is capable of expressing a CAR is aT-cell containing nucleic acid sequences for the expression of the CARin the cell.

As used herein, a “costimulatory element” or “costimulatory signalingdomain” or “costimulatory polypeptide” are defined to be theintracellular portion of a costimulatory polypeptide. A costimulatorypolypeptide can be represented in the following protein families: TNFreceptor proteins, Immunoglobulin-like proteins, cytokine receptors,integrins, signaling lymphocytic activation molecules (SLAM proteins),and activating natural killer cell receptors. Examples of suchpolypeptides include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40,ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1),CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, MyD88, and thelike.

As used herein, a “Cmax” is defined to mean the maximum concentration ofa polypeptide produced by a cell after the cell is stimulated oractivated to produce the polypeptide.

As used herein, a “cytokine C_(max)” is defined to mean the maximumconcentration of cytokine produced by an immune cell after stimulationor activation to produce the cytokine.

As used herein, a “cytotoxic polypeptide C_(max)” is defined to mean themaximum concentration of cytotoxic polypeptide produced by an immunecell after stimulation or activation to produce the cytotoxicpolypeptide.

As used herein, an “effective amount” or “therapeutically effectiveamount” are used interchangeably, and defined to be an amount of acompound, formulation, material, or composition, as described hereineffective to achieve a particular biological result.

As used herein, an “epitope” is defined to be the portion of an antigencapable of eliciting an immune response, or the portion of an antigenthat binds to an antibody. Epitopes can be a protein sequence orsubsequence that is recognized by an antibody.

As used herein, an “expression vector” and an “expression construct” areused interchangeably, and are both defined to be a plasmid, virus, orother nucleic acid designed for protein expression in a cell. The vectoror construct is used to introduce a gene into a host cell whereby thevector will interact with polymerases in the cell to express the proteinencoded in the vector/construct. The expression vector and/or expressionconstruct may exist in the cell extrachromosomally or integrated intothe chromosome. When integrated into the chromosome the nucleic acidscomprising the expression vector or expression construct will be anexpression vector or expression construct.

As used herein, an “extracellular element” is defined as the antigenbinding or recognition element of a Chimeric Antigen Receptor.

As used herein, a “hematopoietic cell” is defined to be a cell thatarises from a hematopoietic stem cell. This includes but is not limitedto myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes,erythrocytes, mast cells, myeloblasts, basophils, neutrophils,eosinophils, macrophages, thrombocytes, monocytes, natural killer cells,T lymphocytes, B lymphocytes and plasma cells.

As used herein, “heterologous” is defined to mean the nucleic acidand/or polypeptide are not homologous to the host cell. For example, aconstruct is heterologous to a host cell if it contains some homologoussequences arranged in a manner not found in the host cell and/or theconstruct contains some heterologous sequences not found in the hostcell.

As used herein “hnRNPLL” is defined to mean heterogeneous nuclearribonucleoprotein L like.

As used herein, an “intracellular element” is defined as the portion ofa Chimeric Antigen Receptor that resides on the cytoplasmic side of theeukaryotic cell's cytoplasmic membrane, and transmits a signal into theeukaryotic cell. The “intracellular signaling element” is that portionof the intracellular element which transduces the effector functionsignal which directs the eukaryotic cell to perform a specializedfunction.

As used herein, a “knottin” or “inhibitor cystine-knots” or“cysteine-knot” are a structural family of ultra-stable peptides withdiverse functions. Knottins contain three disulfide bonds connected in aparticular arrangement that endows these peptides with high thermal,proteolytic, and chemical stability. Knottins have a core scaffoldstructure with peptide loops that can confer binding specificity to thepeptide. Knottins have been engineered to introduce diversity into theloops to create binding specificity for desired targets.

As used herein, “RNA destabilizing element” or “RDE” are usedinterchangeably and both are defined as a nucleic acid sequence in anRNA that is bound by proteins and which protein binding changes thestability and/or translation of the RNA. Examples of RDEs include ClassI AU rich elements (ARE), Class II ARE, Class III ARE, U rich elements,GU rich elements, and stem-loop destabilizing elements (SLDE). Withoutwishing to be bound by theory, RDE's may also bind RNA stabilizingpolypeptides like HuR.

As used herein, a “single chain antibody” (scFv) is defined as animmunoglobulin molecule with function in antigen-binding activities. Anantibody in scFv (single chain fragment variable) format consists ofvariable regions of heavy (VH) and light (VL) chains, which are joinedtogether by a flexible peptide linker.

As used herein, a “T-lymphocyte” or T-cell” is defined to be ahematopoietic cell that normally develops in the thymus. T-lymphocytesor T-cells include, but are not limited to, natural killer T cells,regulatory T cells, helper T cells, cytotoxic T cells, memory T cells,gamma delta T cells and mucosal invariant T cells.

As used herein, “Tox” is defined to mean thymocyte selection associatedhigh mobility group box.

As used herein, “transfected” or “transformed” or “transduced” aredefined to be a process by which exogenous nucleic acid is transferredor introduced into a host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

As used herein, a “transmembrane element” is defined as the elementbetween the extracellular element and the intracellular element. Aportion of the transmembrane element exists within the cell membrane.

Combination Therapies

This disclosure provides compositions and methods for providing a CART-lymphocyte expressing a transgene under the control of an RDE incombination or in an order of succession with another therapy. The othertherapy can include, for example, a chemotherapeutic, an antibody, andantibody-drug conjugate, a radiotherapy, an alkylating agent, a plantalkaloid, an antitumor antibiotic, an antimetabolite, a topoisomeraseinhibitor, and/or an anti-neoplastic. For example, the other therapy canbe an antibody drug conjugate that has the same or different specificityas the CAR T-lymphocyte.

Antibodies and antibody-drug conjugates (ADC) can bind to a tumorassociated antigen, including, for example, any of the tumor associateantigens described herein as targets for a CAR. The drug component ofthe ADC can be, for example, a chemotherapeutic, a radionucleotide, analkylating agent, a plant alkaloid, an antitumor antibiotic, anantimetabolite, a topoisomerase inhibitor, and/or an anti-neoplastic.The drug component of the ADC can be attached to the antibody through alinker which can be cleavable or non-cleavable in nature.

Alkylating agents can include, for example, mustard gas derivatives(e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan, orifosfamide), ethylenimines (e.g., thiotepa or hexamethylmelamine),alkylsulfonates (e.g., busulfan), hydrazines and triazines (e.g.,altretamine, procarbazine, dacarbazine, or temozolomide), nitrosoureas(e.g., carmustine, lomustine or streptozocin), and metal salts (e.g.,carboplatin, cisplatin, or oxaliplatin). Plant alkaloids can include,for example, Vinca alkaloids (e.g., vincristine, vinblastine, orvinorelbine), taxanes (e.g., paclitaxel or docetaxel), podophyllotoxins(e.g., etoposide or tenisopide), and camptothecan analogs (e.g.,irinotecan or topotecan). Antitumor antibiotics can include, forexample, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin,mixoantrone, or idarubicin), and chromomycins (e.g., dactinomycin orplicamycin). Antimetabolites can include, for example, folic acidantagonists (e.g., methotrexate), pyrimidine antagonists (e.g.,5-flurouracil, foxuridine, cytarabine, capecitabine, or gemcitabine),purine antagonists (e.g., 6-mercaptopurine or 6-thioguanine), andadenosine deaminase inhibitors (e.g., cladribine, fludarabine,nelarabine, or pentostatin). Topoisomerase inhibitors can include, forexample, topoisomerase I inhibitors (e.g., irinotecan or topotecan) andtopoisomerase II inhibitors (e.g., amsacrine, etoposide, etoposidephosphate, or teniposide). Anti-neoplastics can include, for example,ribonucleotide reductase inhibitors (e.g., hydroxyurea), adrenocorticalsteroid inhibitors (e.g., mitotane), enzymes (e.g., asparaginase orpegaspargase), antimicrotubule agents (e.g., estramustine), andretinoids (e.g., bexarotene, isotretinoin, or tretinoin).

The drug component can also be an anthracycline, a camptothecin, atubulin inhibitor, a maytansinoid, a calicheamycin, apyrrolobenzodiazepine dimer (PBD), an auristatin, a nitrogen mustard, anethylenimine derivative, an alkyl sulfonate, a nitrosourea, a triazene,a folic acid analog, a taxane, a COX-2 inhibitor, a pyrimidine analog, apurine analog, an antibiotic, an enzyme inhibitor, anepipodophyllotoxin, a platinum coordination complex, a vinca alkaloid, asubstituted urea, a methyl hydrazine derivative, an adrenocorticalsuppressant, a hormone antagonist, an antimetabolite, an alkylatingagent, an antimitotic, an anti-angiogenic agent, a tyrosine kinaseinhibitor, an mTOR inhibitor, a heat shock protein (HSP90) inhibitor, aproteosome inhibitor, an HDAC inhibitor, a pro-apoptotic agent, and acombination thereof.

Specific drugs of use may be selected from the group consisting of5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin,2-pyrrolinodoxorubicine (2-PDox), a pro-drug form of 2-PDox(pro-2-PDox), cyano-morpholino doxorubicin, doxorubicin glucuronide,endostatin, epirubicin glucuronide, erlotinib, estramustine,epidophyllotoxin, erlotinib, entinostat, estrogen receptor bindingagents, etoposide (VP16), etoposide glucuronide, etoposide phosphate,exemestane, fingolimod, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR(FUdR-dO), fludarabine, flutamide, farnesyl-protein transferaseinhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101,gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,ifosfamide, imatinib, lapatinib, lenolidamide, leucovorin, LFM-A13,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,monomethylauristatin F (MMAF), monomethylauristatin D (MMAD),monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, SN-38, sorafenib,streptozocin, SU11248, sunitinib, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839. Preferably, the drug is SN-38.

In an aspect the combination therapy is a protein conjugate. The proteinconjugate can carry a payload that can be a therapeutic, diagnostic, ora reporter. A single molecule of the therapeutic, diagnostic or reportermay be present or two or more molecules may be present. The therapeuticcan be a chemotherapeutic including, for example, any of those describedherein such as a radionucleotide, an alkylating agent, a plant alkaloid,an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor,and/or an anti-neoplastic. The payload of the conjugate can be any oneor more of these therapeutics, diagnostics and/or reporters. The proteincan be a fragment, a monomer, a dimer, or a multimeric protein. Theprotein can be an antibody, an antibody fragment or derivative, a singlechain antibody, an enzyme, cytokine, chemokine, receptor, blood factor,peptide hormone, toxin, and/or transcription factor.

Many conjugating reagents can be used to conjugate a payload to aprotein. Such reagents may contain at least one functional group capableof reacting with a protein or peptide. For example, the conjugatingreagent may comprise a functional group capable of reacting with atleast one electrophile or, especially, nucleophile, present in theprotein, the functional group being attached to the payload via thelinker. Any type of known conjugation reaction may be used to form theconjugate. For example, the reaction can be carried out using the knownmethods of thiol bonding, amine conjugation, or click chemistry. Thereagent may contain a maleimide group, an N-hydroxysuccinimide group, aclick-chemistry group, for example an azide or alkyne group, an aminegroup, a carboxyl group, a carbonyl group, or an active ester group.Other possible approaches include the use of proteins that have beenrecombinantly engineered with an amino acid specifically for conjugationsuch as engineered cysteines or non-natural amino acids, and enzymaticconjugation through a specific enzymatic reaction such as withtransglutaminase. The reaction site on the protein may be eithernucleophilic or electrophilic in nature. Common protein conjugationsites are at lysine or cysteine amino acid residues or carbohydratemoieties. Alternatively, conjugation may occur at a polyhistidine tagwhich has been attached to a binding protein.

A conjugating reagent can be advantageously capable of reacting with anucleophile in a protein and hence becoming chemically bonded thereto.In these examples, the conjugating reagent typically includes at leastone leaving group which is lost on reaction with a nucleophile. Theconjugating reagent may, for example, include two or more leavinggroups. The conjugating reagent can be capable of reacting with twonucleophiles. The conjugating reagent can comprise at least two leavinggroups. When two or more leaving groups are present, these may be thesame or different. Alternatively, a conjugating reagent may contain asingle group which is chemically equivalent to two leaving groups andwhich single group is capable of reacting with two nucleophiles.Nucleophilic groups include, for example, sulfur atoms and amine groups,and nucleophilic groups in proteins are for example provided bycysteine, lysine or histidine residues. Nucleophilic groups can be asulfur atom present in a cysteine residue of a protein. Such structuresmay be obtained by reduction of a disulfide bond in the protein. Thenucleophilic group may be an imidazole group in a histidine residue ofthe protein, e.g., as present in a polyhistidine tag.

The conjugates can contain a linker which connects the therapeutic,diagnostic or labelling agent to the protein or peptide in theconjugate. The backbone of the linker can be a continuous chain of atomswhich runs from the therapeutic, diagnostic or labelling agent at oneend to the protein or peptide at the other end. The linker may contain adegradable group, i.e. it may contain a group which breaks underphysiological conditions, separating the payload from the protein towhich it is, or will be, bonded. Alternatively, the linker is notcleavable under physiological conditions. Where a linker breaks underphysiological conditions, it is preferably cleavable under intracellularconditions. Where the target is intracellular, preferably the linker issubstantially insensitive to extracellular conditions (i.e. so thatdelivery to the intracellular target of a sufficient dose of thetherapeutic agent is not prohibited).

Where the linker contains a degradable group, this is generallysensitive to hydrolytic conditions, for example it may be a group whichdegrades at certain pH values (e.g. acidic conditions).Hydrolytic/acidic conditions may for example be found in endosomes orlysosomes. Examples of groups susceptible to hydrolysis under acidicconditions include hydrazones, semicarbazones, thiosemicarbazones,cis-aconitic amides, orthoesters and ketals. The degradable linker canalso be an acid-cleavable linker or a reducible linker. The reduciblelinker may comprise a disulfide group. The linker may also contain agroup which is susceptible to enzymatic degradation, for example it maybe susceptible to cleavage by a protease (e.g. a lysosomal or endosomalprotease) or peptidase. For example, it may contain a peptidyl groupcomprising at least one, for example at least two, or at least threeamino acid residues (e.g. Phe-Leu, Gly-Phe-Leu-Gly, Val-Ala, Val-Cit,Phe-Lys, Glu-Glu-Glu). For example, it may include an amino acid chainhaving from 1 to 5, for example 2 to 4, amino acids. The enzymecleavable linker can also comprise a chemical group which can be cleavedor degraded by one or more lysosomal enzymes. Suitable groups include,for example, a valine-citrulline dipeptide group, a phenylalanine-lysinedipeptide group, and a β-glucuronide group.

When the protein in the protein conjugate is an antibody (e.g., fulllength, fragment, and/or single chain) one end of the first linker canbe covalently attached to the antibody. The antibody-reactive end of thelinker can be a site that is capable of conjugation to the antibodythrough a cysteine thiol or lysine amine group on the antibody, and socan be a thiol-reactive group such as a double bond (as in maleimide) ora leaving group such as a chloro, bromo, or iodo, or an R-sulfanylgroup, or an amine-reactive group such as a carboxyl group.

The CAR therapy (e.g., with a GOLD-controlled transgene) and the othertherapy can be provided to a subject at the same time, or one can beprovided to the subject before the other, or the CAR therapy and theother therapy can be provided in alternating cycles, or the CAR therapytogether with the other therapy can be provided in cycles, or othercombinations of administration can be used. The CAR therapy can becombined with an antibody conjugate (ADC) therapy where the CAR and theADC bind to the same antigen or bind to different antigens. When the CARand ADC bind to the same antigen, the CAR and the ADC can bind to thesame or different epitopes on the antigen. One of the ADC or the CARtherapy can be provided to the subject first, and followed by the otherafter a period of treatment with the first. The ADC, either alone or incombination with another approved therapy (e.g. chemotherapy and/orimmume checkpoint inhibitors) can be provided to the subject first toreduce the tumor burden in the subject prior to the administration ofthe CAR therapy. Alternatively, the ADC and CAR therapy can be providedto the subject at the same time. Or the CAR therapy can be providedfirst followed by the ADC therapy.

RNA Destabilizing Elements

RNA destabilizing elements (RDE) are nucleic acids that affect ormaintain the stability of an RNA molecule or the translation kinetics ofan RNA molecule. Some RDEs are bound by polypeptides which destabilize(e.g., cleave) the RNA, or prevent translation, leading to loss offunction for the RNA. Some RDE binding polypeptide stabilizes the RNAincreasing the half-life of the RNA. RDEs can be used to control theexpression of a transgene, e.g., a transgene encoding a chimeric antigenreceptors. RDEs can be used with RNA control devices, DEs, and/or SideCARs to regulate the expression of a transgene. The RDEs can also beused to control expression of transgenes encoding polypeptides otherthan a CAR. Other transgenes may encode, for example, a cytokine, anantibody, a checkpoint inhibitor, a granzyme, an apoptosis inducer,complement, a cytotoxic small molecule, other cytotoxic compounds, apolypeptide for imaging, or other polypeptide that can have a desiredeffect. The RDE can control the delivery of a transgene payload.Examples of RDEs include, for example, AU rich elements, U richelements, GU rich elements, and certain stem-loop elements. ExemplaryRDEs are described in Kovarik et al., Cytokine 89:21-26 (2017); Ray etal., Nature 499:172-177 (2013); Castello et al., Cell 149:1393-1406(2012); Vlasova et al., Molc. Cell. 29:263-270 (2008); Barreau et al.,Nucl. Acids Res. vol 33, doi:10.1093/nar/gki1012 (2006); Meisner et al.,ChemBioChem 5:1432-1447 (2004); Guhaniyogi et al., Gene 265:11-23(2001), all of which are incorporated by reference in their entirety forall purposes.

The RDE can be a Class I AU rich element (dispersed AUUUA (SEQ ID NO:1)in U rich context), a Class II AU rich element (overlapping(AUUUA)_(n)), a Class III AU rich element (U-rich stretch), a stem-loopdestabilizing element (SLDE), a cytokine 3′ UTR (e.g., INF-γ, IL-2,T-cell receptor α chain, TNFα, IL-6, IL-8, GM-CSF, G-CSF etc.), and asequence of AUUUAUUUAUUUA (SEQ ID NO: 2). Khabar, WIREs RNA 2016, doi:10.1002/wrna.1368 (2016); Palanisamy et al, J. Dent. Res. 91:651-658(2012), both of which are incorporated by reference in their entiretyfor all purposes. The RDE can also be a GU rich element comprised of oneor more of, for example, UUGUU (SEQ ID NO: 3), UGGGGAU (SEQ ID NO: 4),or GUUUG (SEQ ID NO: 5). The RDE can be a U-rich element comprised ofone or more of, for example, UUUGUUU (SEQ ID NO: 6), U (SEQ ID NO: 7),UUUAUUU (SEQ ID NO: 8), UUUGUUU (SEQ ID NO: 9), UUAGA (SEQ ID NO: 10),or AGUUU (SEQ ID NO: 11). In some aspects, multiple RDEs can be combinedto make a regulatory unit, for example, multiple RDEs that have the samesequence can be arranged in a concatemer or can be arranged withintervening sequence in between some or all of the RDEs. The RDEsequence can be modified to increase or decrease the affinity of an RNAbinding protein(s) for the RDE. For example, an AU rich RDE can bechanged to alter the affinity of glyceraldehyde phosphate dehydrogenase(GAPDH) to the RDE. This change in affinity can alter theGAPDH-activation threshold for expression of a transgene regulated bythe RDE to which GAPDH binds.

The disclosure assigns AU# designations to some RDEs and these RDEs canbe referred to by the AU# or the gene name from which the RDE isderived. Some AU#s and the corresponding gene from which the RDE isderived include, for example, AU 1 (CD40LG), AU 2 (CSF2), AU 3 (CD247),AU 4 (CTLA4), AU 5 (EDN1), AU 6 (IL2RA), AU 7 (SLC2A1), AU 8 (TRAC), AU9 (CD274), AU 10 (Myc), AU 11 (CD19), AU 12 (IL4), AU 13 (IL5), AU 14(IL6), AU 15 (IL9), AU 16 (IL10), AU 17 (IL13), AU 18 (FOXP3), AU 19(TMEM-219), AU 20 (TMEM-219snp), AU 21 (CCR7), AU 22 (SEM-A4D), AU 23(CDC42-SE2), AU 24 (CD8), AU 27 (bGH), and AU 101 (IFNg).

The RDE can be from the 3′ UTR of a gene encoding, for example, IL-1,IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, GM-CSF, G-CSF, VEGF, PGE₂, COX-2,MMP (matrix metalloproteinases), bFGF, c-myc, c-fos, betal-AR, PTH,interferon-gamma, MyoD, p21, Cyclin A, Cyclin B1, Cyclin D1, PAI-2, NOSHANOS, TNF-alpha, interferon-alpha, bc1-2, interferon-beta, c-jun,GLUT1, p53, Myogenin, NF-M, or GAP-43, lymphocyte antigen 96, SUPV3L1,SFtPA2, BLOC1S2, OR10A6, OR8D1, TRPT1,CIP29, EP400, PLE2, H3ST3A1,ZNF571, PPP1R14A, SPAG4L, OR10A6 and KIR3DL. Other RDEs are found in,for example, the 3′-UTRs from GLMN, AMY2B, AMY2A, AMY2A, AMY1A, TRIM33,TRIM33, TRIM33, CSRP1, PPP1R12B, KCNH1, Reticulon 4, MRPL30, Nav1.2,Tissue_factor_pathway_inhibitor, EEF1B2, CRYGB, ARMC9, RPL15, EAF2,MRPS22, MRPS22, COPB2, PDCD10, RE1-silencing_transcription_factor,Amphiregulin, AP1AR, TLR3, SKP2,Peptidylglycine_alpha-amidating_monooxygenase, TNFAIP8, Interleukin 9,PCDHA2, PCDHA12, Aldehyde_dehydrogenase_5_family,_member_A1, KCNQ5,COX7A2, Monocarboxylate_transporter_10, MLLT4, PHF10, PTPN12,MRNA_(guanine-N7-)-methyltransferase, WHSC1L1,Tricho-rhino-phalangeal_syndrome_Type_1, Interferon_alpha-1, ZCCHC6,Retinitis_pigmentosa_GTPase_regulator, MED14, CLCN5, DNA2L, OR52D1,NELL1, SLC22A25, SLC22A10, TRPC6, CACNA2D4, EPS8, CT2 (gene),Mitochondrial_ribosomal_protein_L42, TAOK3, NUPL1,Endothelin_receptor_type_B,Survival_of_motor_neuron_protein-interacting_protein 1, POLE2,Hepatic_lipase, TPSG1, TRAP1, RPS15A, HS3ST3A1, CROP_(gene),Apolipoprotein_H, GRB2, CEP76, VPS4B, Interleukin 28B, IZUMO1, FGF21,PPP1R15A, LIN7B, hnRNPLL, Tox, and CDC45-related_protein.

Still other RDEs can be found in, for example, the 3′UTRs of SCFD1,MAL2, KHSRP, IQCB1, CAMP_responsive_element_modulator, MFAP5, SBF2,FKBP2, PDCD10, UBE2V2, NDUFAB1, Coiled-Coil_Domain_Containing_Protein,ALG13, TPTE, Enaptin, Thymopoietin, Delta-like 1, C11orf30,Actinin_alpha_4, TMEM59, SP110, Dicer, TARDBP, IFNA17, IFNA16, IFNA14,ZMYM3, Interleukin_9,_type_I, OPN1SW, THSD1, ERGIC2, CAMK2B, WDR8, FXR1,Thymine-DNA_glycosylase, Parathyroid_hormone-related_protein, OSBPL3,Ran, GYPE, AKAP4, LOC642658, L2HGDH, AKAP1, Zinc_finger_protein_334,TC2N, FKBPL, GRB14, CXorf67, CXorf66, CEP76, Gastricsin, CEP70, CYP26A1,NAA35, Aryl_hydrocarbon_receptor_nuclear_translocator, KLC4, GPR112,LARP4, NOVA1, UBE2D3, ITGA6, GPR18, MGST_type_A,RE1-silencing_transcription_factor, ASPM, ZNF452, KIR2DS4, AHSA1, TMTC4,VSX1, P16, MRPL19, CCL20, TRPT1, Hepatic_lipase, PDLIM5,CCDCl53,′CCDCl55, GAPVD1, HOXB2, KCNQ5, BRCC3, GTF2IRD1, CDK5RAP3,Transcription_factor_II_B, ZEB1, IRGM, SLC39A6, RHEB, PSIP1, RPS6KA5,Urokinase_receptor, GFM1, DNAJC7, Phosphoinositide-dependent_kinase-1,LMOD3, TTC35, RRP12, ATXN2, ACSM3, SOAT1, FGF8, HNRPH3, CTAGE5, POLG2,DYRK3, POLK, Cyclin-dependent_kinase_inhibitor_1C, CD137, Calmodulin_1,ZNF571, CNOT2, CRYZL1, SMC3, SMC4, SLC36A1, Decorin, HKR1, ERC1, S100A6,RIMS1, TMEM67, Mitochondrial_ribosomal_protein_L42, MECP2, RNF111,SULT1A1, MYLK3, TINAG, PRKAR1A, RGPD5, UBE2V1, SAR1B, SLC27A6, ZNF638,RAB33A, TRIOBP, MUCL1, CADPS2, MCF2L, TBCA, SLC17A3, LEO1, IFNA21,RUNX1T1, PRKD2, ATP11B, MORC2, RBM6, KLRD1, MED31, PPHLN1, HMGB2,DNA_repair_and_recombination_protein_RAD54-like, RBM9′, ARL11, HuD,SPEF2, CBLL1, SLC38A1, ‘Caspase 1’, S100G, CA1_, CELA1, PTS, ITM2B,Natriuretic_peptide_precursor_C, TRPP3, IMPDH2, DPYS, CDCA3, EFCAB6,SLIT2, SIPA1L1, FIP1L1, ATP6V1B2, HSD17B4, HSD17B7, NDUFC1, CROP, CD48,APPBP1, CD44, CD46, Histone_deacetylase_2_type_XI, Interleukin 4,Tricho-rhino-phalangeal_syndrome_Type_1, SEC61G, TR1P12, PLEKHO1,SEC61B, ST6GALNAC1, CPVL, E2F7, UTP20, E2F5, PARD3, EXOC7, HEXB,Caspase_recruitment_domain-containing_protein 8, MBD4, PPP4C, Helicase,Phosducin, SPG11, CGGBP1, PSKH1, Cathepsin_S, orexin, IMMP2L, C2orf28,Laminin, EIF3S6, LRRC41_type_XII, Cathepsin_C, HPS6, ARAF,Zinc_finger_and_BTB_domain-containing_protein_16,Sex_hormone-binding_globulin, FBLN2, Suppressor_of_cytokine_signaling_1,TMEM0126A, DOM3Z, TSFM POLQ-like, DYNLT3, CDH9, EAF2, MIPEP, NDUFA12,HDAC8, MKKS, FGG, IL36G, CDCA7, CRISPLD2, Olfactomedin-like_2b, MRPL32,MRPL33, AHI1, SMARCAL1, UTP14A, SSH2, Dystonin, Contactin_6, PPFIBP1,THOC1, CNOT1, RHCE, SLC41A3, SLC2A9, SNAP23, RFX3, GNG4, MRPL40, LSR,Angiogenin, TRIP4, VRK1, COUP-TFII, FOXP2, SNX2, Nucleoporin_85, RPL37A,RPL27A, SEC62, Calcium-activated_potassium_channel_subunit_alpha-1,SMARCE1, RPL17, CEP104, CEP290, VPS29, ANXA4, Zinc_finger_protein 737,DDX59, SAP30, NEK3, Exosome_component_9,Receptor_for_activated_C_kinase_1, Peptidylprolyl_isomerase_A, TINP1,CEACAM1, DISC1, LRRTM1, POP1_Lamin_B1,SREBP_cleavage-activating_protein, COX6C, TLR_1, ARID2, LACTB, MMS22L,UBE2E3, DAP3, ZNF23, SKP2, GPR113, IRF9 Ghrelin O-acyltransferase,NEIL3, EEF1E1, COX17, ESD_, Dentin_sialophosphoprotein, HDAC9, RFC4,CYLD, RPLP0, EIF2B3, UGT2A1, FABP7, TRIP11, PLA2G4A, AKR1C3, INTS12,MYH1, ZBTB17, MYH4, NLRP2, MECOM, MYH8, Thermogenin_receptor_2, IFI16,THYN1, RAB17, ETFA, Cystic_fibrosis_transmembrane_conductance_regulator,F13B, RAB6A, ST8SIA1, SATB2, SATB1, HMG20B, UHRF1, CNOT3, ProstaglandinEP2 receptor, FAM65B, Peroxisome_proliferator-activated_receptor_gamma,KvLQT2, GRIK5, SHOC2, Cortactin, FANCI, KIAA1199, Kynureninase,Decoy_receptor_1, NEU3, PHF10, Methyl-CpG-binding_domain_protein 2,RABGAP1, CEP55, SF3B1, MSH5, MSH6, CREB-binding_protein, LIMS1, SLC5A4,CCNB1IP1, RNF34, SORBS2, UIMC1, SOX5, YWHAZ, ICOSLG, NOP58,Zinc_finger_protein 679, PHKB, MED13, ABCB7, COQ9, C14orf104,Zinc_finger_protein 530, KLRC2, LSM8, NBR1, PRKCD,Long-chain-aldehyde_dehydrogenase, MTSS1, Somatostatin,Ubiquitin_carboxyl-terminal_hydrolase_L5, WDR72, FERMT3, Nuclearreceptor related-1_protein, Citrate_synthase, VPS11, KIZ, ZFYVE27,BCKDHB, Hypocretin, CACNG2, PTCH1, Carbonic_anhydrase_4,Nucleoporin_107, LDL_receptor, LEKTI, FBXO11, NDUFB3, FCHO2, CEP78,RAPGEF6, PPIL3, NIN, RAPGEF2, Growth_hormone_1, Growth_hormone_2, MNAT1,Nav1, MAP3K8, SUGT1, LAIR1, Hyaluronan-mediated_motility_receptor,MAP3K2, MPP2, TFB2M, CRB3, MPP5, CACNA1G, DLGAP2, INHBA, MAGI2, CIP29,SETDB1, Cytochrome_b5, TRPV2, Interleukin_1_receptor, HOXD8, TIMM10,ATXN2L, CLCN2, CREB1, TNIP1, CBLB, Factor_V, USP33, SON, RBBP8,SLC22A18, PTPN12, ADCY8, MYLK, KIF23, REXO2, BST1, TOP3B, COPB1, AXIN2,COPB2, TNRC6B, Guanidinoacetate N-methyltransferase,Acyl-CoA_thioesterase_9, C4orf21, TSHB, FRS3, EPB41, Cyclin_T2, LAIR2,Nucleoporin_43, APLP2, TNFRSF19, Death-associated_protein_6,Epithelial_cell_adhesion_molecule, CLEC7A, Gephyrin, CLDND1, VPS37A,PCDHAC2, Bone_morphogenetic_protein_4, NVL, RBM33, RNF139,Sperm_associated_antigen_5, PLCB1,Glial_cell_line-derived_neurotrophic_factor, PARP4, PARP1, MAN2A1,Bone_morphogenetic_protein 1, PAX4, BCCIP, MMPI, Decoy_receptor_3,RAMP2, NCAPD3, LRRC37A, RWDD3, UBE2A, UBE2C, SLC3A1, MRPS22, CDC14A,ITSN1, POLE2, MYC-induced_nuclear_antigen, TMLHE,Glutamate_carboxypeptidase_II, GPR177, PPP2R5C, KIAA1333, RPP38, MYO1F,Farnesoid_X_receptor, Caldesmon, FBXO4, FBXO5, OPN1MW, PIGN, ARNTL2,BCAS3, C6orf58, PHTF2, SEC23A, NUFIP2, OAZ1, Osteoprotegerin, ANAPC4,ATP6V0A2, SPAM1, PSMA6, TAS2R30, RABEP1, DPM3, SLC6A15, RPS26, RPS27,RPS24, RPS20, RPS21, ARHGAP24, Catechol-O-methyl transferase, ERCC5,Transcription_initiation_protein_SPT3 homolog, OR1E1, ZNRF1, GMEB1,CCT2_GNAQ, Mucin_6, Mucin_4, LRP5, PDE9A, C2orf3, EZH2,Epidermal_growth_factor_receptor, TMTC2, PDE4A, EPH_receptor_A4, PPIB,DENND4A, ANTXR1, ANTXR2, Nucleoporin_88, SLCO1B3, COG8, RBMS1, MAP7,HIST2H2BE, AEBP2, DCLRE1A, RPL24, HNRPA2B1, RPL21, RPL23, MAPKAP1,NIPBL, ATG7, SERPINI2, GYLTL1B, ATP5G2, DIP2A, AMY2A, CEP63, TDRD7,PIEZO1, CLDN20, GRXCR1, PMEL, NIF3L1, MCC_, PCNX, TMBIM4, DUSP12,ZMYND8, GOSR1, Interferon_gamma_receptor_1, LDB3, PON3, C1D, ABCC8,COQ7, COQ6, AMELY, HAVCR1, PICALM, Sjogren_syndrome_antigen_B, PLK4,HBB, AKT1, PCDHGB7, C6orf10, UBR1, Retinoblastoma-like_protein 1, GRK6,WWC2, GRK4, INPP4B, SLC34A1, GOLGA2, MYCBP2, PTP4A2, NUCB2, MAGOH,RPP40, Alpha-2A_adrenergic_receptor, SPAG11B, Nucleoporin_205, COG1,Motile_sperm_domain_containing_3, KCNMB3,Motile_sperm_domain_containing_1, KLHL7, KCNN2, TSPAN8, GPR21,Translocator_protein, HNRNPLL, ABHD5, CAB39L, Amphiregulin, GPR1,Interleukin_18, EIF4G3, Interleukin_15, CCDCl80, CD2AP, NFS1, GRB2,ULBP2, Vascular_endothelial_growth_factor_C, RPS3, TLR8,BCL2-related_protein_A1, RHOT1, Collagen, Centromere_protein E, STMN2,HESX1, RPL7, Kalirin, PCMT1, HLA-F, SUMO2, NOX3, EP400, DNM3, EED,NGLY1, NPRL2, PLAC1, Baculoviral_IAP_repeat-containing_protein_3,C7orf31, TUBA1C, HAUS3, IFNA10, MYST4, DCHS1, SIRT4, EFEMP1, ARPC2,MED30, IFT74, PAK1IP1, DYNC1LI2, POLR2B, POLR2H, KIF3A, PRDM16, PLSCR5,PEX5, Parathyroid_hormone_1_receptor, CDC23, RBPMS, MAST1, NRD1, BAT5,BAT2, Dock11, GCSH, POF1B, USP15, POT1, MUTYH, CYP2E1, FAM122C,A1_polypeptide, Flavin_containing_monooxygenase_3, HPGD, LGALS13,MTHFD2L, Survival_motor_neuron_domain_containing_1, PSMA3, MRPS35,MHC_class_I_polypeptide-related_sequence_A, SGCE, REPS1, PPP1R12A,PPP1R12B, PABPC1, MAPK8, PDCD5, Phosphoglucomutase_3, Ubiquitin_C,GABPB2, Mitochondrial_translational_release_factor_1, PFDN4, NUB1,SLC13A3, ZFP36L1, Galectin-3, CC2D2A, GCA,Tissue_factor_pathway_inhibitor, UCKL1, ITFG3, SOS1, WWTR1, GPR84,HSPA14, GJC3, TCF7L1, Matrix_metallopeptidase_12, ISG20, LILRA3,Serum_albumin, Phosducin-like, RPS13, UTP6, HP1BP3, IL12A,HtrA_serine_peptidase_2, LATS1, BMF_, Thymosin_beta-4, B-cell_linker,BCL2L11, Coagulation_factor_XIII, BCL2L12, PRPF19, SFRS5,Interleukin_23_subunit_alpha, NRAP, 60S_ribosomal_protein_L14, C9orf64,Testin, VPS13A, DGKD, PTPRB, ATP5C1, KCNJ16, KARS, GTF2H2, AMBN, USP13,ADAMTSL1, TRO_, RTF1, ATP6V1C2, SSBP1,SNRPN_upstream_reading_frame_protein, RPS29, SNRPG, ABCC10, PTPRU,APPL1, TINF2, TMEM22, UNC45A, RPL30, PCDH7, Galactosamine-6 sulfatase,UPF3A, ACTL6A, ACTL6B, IL3RA, SDHB, Cathepsin L2, TAS2R7, Cathepsin_L1,Pituitary_adenylate_cyclase-activating_peptide, RPN2, DYNLL1, KLK13,NDUFB3, PRPF8, SPINT2, AHSA1, Glutamate_carboxypeptidase_II, DRAP1,RNASE1, Olfactomedin-like_2b, VRK1, IKK2, ERGIC2, TAS2R16, CAMK2G,CAMK2B, Estrogen_receptor_beta, NADH_dehydrogenase, RPL19, NUCB2,KCTD13, ubiquinone, H2AFY, CEP290, PABPC1, HLA-F, DHX38, KIAA0922,MPHOSPH8, DDX59, MD32, ZBP1, C16orf4, UACA, C6orf142, MRPL39,Cyclin-dependent_kinase_7, Far_upstream_element-binding_protein_1,SGOL1, GTF2IRD1, ATG10, Dermcidin, EPS8L2, Decorin,Nicotinamide_phosphoribosyltransferase, CDCl20, MYB, WNTSA, RBPJ,DEFB103A, RPS15A, ATPSH, RPS3, FABP1, SLC4A8, Serum_amyloid_P_component,ALAS1, MAPK1, PDCD5, SULT1A1, CHRNA3, ATXN10, MNAT1, ALG13, Ataxin_3,LRRC39, ADH7, Delta-sarcoglycan, TACC1, IFNA4,Thymic_stromal_lymphopoietin, LGTN, KIAA1333, MSH6, MYOT, RIPK5,BCL2L11, RPL27, Rnd1, Platelet_factor_4, HSD17B7, LSM8, CEP63, INTS8,CTNS, ASAHL, CELA3A, SMARCAL1, HEXB, SLC16A5, MAP3K12, FRMD6.

Additional RDEs are found in the 3′-UTRs of long noncoding RNAs, orprimary transcripts encoding miRNAs. For example, RDEs from the 3′-UTRof THRIL (linc 1992), NIKILA lncRNA, SeT lncRNA, lncRNAs uc.197,RP11-172E9.2, LINC00598, lncRNAs LOC100128098, RP11-150012.3, and theprimary transcripts encoding miR-146a, miR-let7e, miR-181c, miR-155,miR-125b, and miR-16.

A class of RDEs includes those which are bound by glycolytic enzymessuch as glyceraldehyde phosphate dehydrogenase (GAPDH). This group ofRDEs includes, for example, AU 19 (TMEM-219), AU 20 (TMEM-219snp), AU 21(CCR7), AU 22 (SEM-A4D), and AU 23 (CDCl42-SE2).

The RDE can be a Class I AU rich element that arises from the 3′ UTR ofa gene encoding, for example, c-myc, c-fos, betal-AR, PTH,interferon-gamma, MyoD, p21, Cyclin A, Cyclin B1, Cyclin D1, PAI-2, orNOS HANOS. The RDE can also be a Class II AU rich element and arisesfrom the 3′ UTR of a gene encoding, for example, GM-CSF, TNF-alpha,interferon-alpha, COX-2, IL-2, IL-3, bc1-2, interferon-beta, or VEG-F.The RDE can be a Class III AU rich element that arises from the 3′ UTRof a gene encoding, for example, c-jun, GLUT1, p53, hsp 70, Myogenin,NF-M, or GAP-43. Other RDEs may be obtained from the 3′-UTRs of a T-cellreceptor subunit (α, β, γ, or δ chains), cytotoxicT-lymphocyte-associated antigen 4 (CTLA4), programmed cell death protein(PD-1), Killer-cell Immunoglobulin-like Receptors (KIR), and LymphocyteActivation Gene-3 (LAG3), and other checkpoint inhibitors. Still otherRDEs may be obtained from the 3′-UTRs of senescence-associated secretoryphenotype genes disclosed in Coppe et al., Ann. Rev. Pathol 5:99-118(2010), which is incorporated by reference in its entirety for allpurposes (e.g., see Table 1).

The RDE can be bound by certain polypeptides including, for example, AREpoly(U) binding/degradation factor (AUF-1), tristetraprolin (TTP), humanantigen-related protein (HuR), butyrate response factor 1 (BRF-1),butyrate response factor 2 (BRF-2), T-cell restricted intracellularantigen-1 (TIA-1), TIA-1 related protein (TIAR), CUG triplet repeat, RNAbinding protein 1 (CUGBP-1), CUG triplet repeat, RNA binding protein 2(CUGBP-2), human neuron specific RNA binding protein (Hel-N1, Hel-N2),RNA binding proteins HuA, HuB and HuC, KH-type splicing regulatoryprotein (KSRP), 3-methylglutaconyl-CoA hydratase (AUH), glyceraldehyde3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (Hsp70), heatshock protein 10 (Hsp10), heterogeneous nuclear ribonucleoprotein A1(hnRNP A1), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2),heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3), heterogeneousnuclear ribonucleoprotein C (hnRNP C), heterogeneous nuclearribonucleoprotein L (hnRNP L), Bc1-2 AU-rich element RNA binding protein(TINO), Poly(A) Binding Protein Interacting Protein 2 (PAIP2), IRP1,pyruvate kinase, lactate dehydrogenase, enolase, and aldolase. The RDEbinding protein also can be an enzyme involved in glycolysis orcarbohydrate metabolism, such as, for example, Glyceraldehyde PhosphateDehydrogenase (GAPDH), enolase (ENO1 or ENO3), Phosphoglycerate Kinase(PGK1), Triosephosphate Isomerase (TPI1), Aldolase A (ALDOA),Phosphoglycerate Mutase (PGAM1), Hexokinase (HK-2), or LactateDehydrogenase (LDH). The RDE binding protein can be an enzyme involvedin the Pentose Phosphate Shunt, including for example, Transketolase(TKT) or Triosephosphate Isomerase (TPI1). Additional exemplary RNAbinding proteins are those described in Castello et al., Molc. Cell63:696-710 (2016); Kovarik et al., Cytokine 89:21-26 (2017); Ray et al.,Nature 499:172-177 (2013); Castello et al., Cell 149:1393-1406 (2012);Vlasova et al., Molc. Cell. 29:263-270 (2008); Barreau et al., Nucl.Acids Res. vol 33, doi:10.1093/nar/gki1012 (2006); Meisner et al.,ChemBioChem 5:1432-1447 (2004); Guhaniyogi et al., Gene 265:11-23(2001), all of which are incorporated by reference in their entirety forall purposes.

The RDE binding protein can be TTP which can bind to RDEs including forexample, one or more of UUAUUUAUU (SEQ ID NO: 12) and AUUUA (SEQ ID NO:1), or KSRP which binds AU-rich RDEs, or Auf1 which binds RDEs includingfor example, one or more of UUGA (SEQ ID NO: 13), AGUUU (SEQ ID NO: 11),or GUUUG (SEQ ID NO: 5), or CELF-1 which binds RDEs including forexample, one or more of UUGUU (SEQ ID NO: 3), or HuR which binds RDEsincluding for example, one or more of UUUAUUU (SEQ ID NO: 8), UUUAUUU(SEQ ID NO: 9), or UUUGUUU (SEQ ID NO: 6), or ESRP1 or ESRP2 which bindsRDEs including for example, one or more of UGGGGAU (SEQ ID NO: 14), orELAV which binds RDEs including for example, one or more of UUUGUUU (SEQID NO: 6). The RDE binding protein can be an enzyme involved inglycolysis, including for example, GAPDH which binds AU rich elementsincluding for example, one or more of AUUUA (SEQ ID NO: 1) elements, orENO3/ENO1 which binds RDEs including for example, one or more ofCUGCUGCUG (SEQ ID NO: 15), or ALDOA which binds RDEs including forexample, one or more of AUUGA (SEQ ID NO: 16).

In an aspect, the RDE can be combined with an RNA control device to makethe regulation by the RDE ligand inducible. For example, an RDE can beoperably linked to an RNA control device where ligand binding by the RNAcontrol device activates the regulatory element (e.g., a ribozyme orriboswitch) which inhibits the RDE (e.g., a ribozyme cleaves the RDE soRDE binding proteins no longer bind, or the riboswitch alters secondarystructure). This places transcripts with the RDE and RNA control deviceunder two types of control from the RDE, first the RDE can regulate thetranscript subject to binding of RDE binding proteins as governed byconditions in the cell, and second, the RDE control can be removed byinducing the RNA control device with ligand. When ligand is added, theRNA control device renders the RDE unavailable for binding and RDEregulation is removed. When ligand is removed, new transcripts that aretranscribed can be under the control of the RDE (as the RNA controldevice will not be activated). Alternatively, an RDE can be operablelinked to an RNA control device where ligand binding turns off theregulatory element (e.g., a ribozyme). In this example, the presence ofligand inhibits the RNA control device and transcripts can be regulatedby the RDE. When ligand is removed, the RNA control device renders theRDE unavailable for binding to RDE binding proteins and RDE regulationof the transcript is removed. The RNA control device could also cleave apolynucleotide that binds to the RDE to form a structure (e.g., a helix)that inhibits RDE proteins from binding to the RDE. In this example, theRNA control device can cleave the inhibitory polynucleotide which thendoes not bind or is inhibited for binding to the RDE. This cleavage bythe RNA control device can be stimulated by ligand binding or inhibitedby ligand binding.

Different RDEs have different kinetic parameters such as, for example,different steady expression levels, different T_(max) (time to maximalexpression level), different C_(max) (maximum expression level),different dynamic range (expression/basal expression), different AUC,different kinetics of induction (acceleration of expression rate andvelocity of expression rate), amount of expression, baseline expression,maximal dynamic range (DR_(max)), time to DR_(max), area under the curve(AUC), etc. In addition, these kinetic properties of the RDEs can bealtered by making concatemers of the same RDE, or combining differentRDEs into regulatory units. Placing RDEs under the control of anoperably linked RNA control device can also alter the kinetic propertiesof the RDE, RDE concatemer, or RDE combinations. Also, small moleculesand other molecules that affect the availability of RDE binding proteinsfor binding RDEs can be used to alter the kinetic response of an RDE,RDE concatemer, and/or RDE combinations. The kinetic response of RDEs,RDE concatemers, and/or RDE combinations can be changed using constructsthat express competitive RDEs in a transcript. Such transcripts with oneor more competing RDEs can compete for RDE binding proteins and so alterthe regulation of the desired gene by an RDE, RDE concatemer, and/or RDEcombination. These competitive RDE transcripts can bind to RDE bindingproteins reducing the amount of RDE binding protein available forbinding to the RDE, RDE concatemer, and/or RDE combination. Thus, RDEs,RDE concatemers, and/or RDE combinations can be selected and/or combinedwith other conditions (discussed above) to provide a desired kineticresponse to the expression of a transgene.

Table 2 in Example 20 shows that different RDEs (e.g., AU elements)provided different kinetics of expression. For example, different RDEs(e.g., AU elements) reached maximal induction (maximal dynamic rangealso known as fold induction) at different time points. The RDEs AU 2and AU 101 reached maximal dynamic range (DR_(max)) at day 1 and thenthe dynamic range (DR) decreased showing reduced expression compared tobasal expression. The RDEs AU 5 and AU 21 had a DR_(max) at day 3/4 andthis expression was maintained out to day 8. The RDEs AU 3, AU 7, AU 10,AU 20 and AU 23 had a DR_(max) on day 6 and expression decreased on day8. The RDEs AU 19 and AU 22 had DR_(max) on day 8. The RDEs (e.g., AUelements) also had differences in the amount of expression covering arange of about 5500 fold comparing the expression of AU 7 to AU 10 (seeTable 1). Thus, RDEs (AU elements) can be selected to provide maximalrates of expression at a desired time point and to provide a desiredamount of polypeptide at that time point.

Some RNA binding proteins increase the rate of RNA degradation afterbinding to the RDE. Some RNA binding proteins decrease the rate ofdegradation of the RNA after binding to the RDE. More than one RNAbinding protein binds can bind to an RDE. In some RDE regulatory units,more than one RNA binding protein binds to more than one RDE. Binding ofone or more of the RNA binding proteins to the one or more RDEs canincrease the degradation rate of the RNA. Binding of one or more of theRNA binding proteins can decrease the degradation rate of the RNA. RNAbinding proteins that increase degradation may compete for binding to anRDE with RNA binding proteins that decrease degradation, so that thestability of the RNA is dependent of the relative binding of the two RNAbinding proteins. Other proteins can bind to the RDE binding proteinsand modulate the effect of the RNA binding protein on the RNA with theRDE. Binding of a protein to the RNA binding protein can increases RNAstability or decrease RNA stability. An RNA can have multiple RDEs thatare bound by the proteins HuR and TTP. The HuR protein can stabilize theRNA and the TTP protein can destabilize the RNA. An RNA can have atleast one RDE that interacts with the proteins KSRP, TTP and/or HuR.KSRP can destabilize the RNA and compete for binding with the HuRprotein that can stabilize the RNA. The KSRP protein can bind to the RDEand destabilizes the RNA and the TTP protein can bind to KSRP andprevent degradation of the RNA. Different proteins may be bound to thesame transcript and may have competing effects on degradation andstabilization rates. Different proteins may be bound to the sametranscript and may have cooperative effects on degradation andstabilization rates. Different proteins may be bound to the sametranscript at different times, conferring different effects ondegradation and stabilization.

The RDE can be a Class II AU rich element, and the RNA binding proteincan be GAPDH. The Class II AU rich element bound by GAPDH can beAUUUAUUUAUUUA (SEQ ID NO: 2). The Class II AU rich element and GADPH canbe used to control the expression of a transgene, a CAR, Smart CAR (RNAcontrol device—CAR), DE-CAR (destabilizing element—CAR), Smart-DE-CAR,and/or Side-CAR. The Class II AU rich element and GADPH also can be usedto effect the expression of a transgene and/or a CAR in a T-lymphocyte.The Class II AU rich element and GADPH can be used to effect theexpression of a transgene and/or a CAR in a CD8+ T-lymphocyte. The ClassII AU rich element and GADPH can be used to effect the expression of atransgene and/or a CAR in a CD4+ T-lymphocyte. The Class II AU richelement and GADPH can be used to effect the expression of a transgeneand/or a CAR in a natural killer cell.

The RDE may have microRNA binding sites. The RDE can be engineered toremove one or more of these microRNA binding sites. The removal of themicroRNA binding sites can increase the on expression from a constructwith an RDE by at least 5, 10, 15, 20, 50 or 100 fold. The RDE with themicroRNA sites can be an RDE that is bound by GAPDH. The removal ofmicroRNA sites from the RDE bound by GAPDH can increase the onexpression of a construct with the GAPDH sensitive RDE by at least 5-10fold. This GAPDH control through the RDE can be used to deliver apayload at a target site. The GAPDH control can be tied to activation ofthe eukaryotic cell by a CAR that recognizes an antigen foundpreferentially at the target site.

The RDE can be the 3′-UTR of IL-2 or IFN-γ, and removal of micro-RNAsites can increase the rate of expression and/or the dynamic range ofexpression from a transgene RNA with the RDE. The RDE can be the 3′-UTRof IL-2 and the removed micro-RNA sites can be the MIR-186 sites whichdeletion increases the kinetics of expression and increases the dynamicrange of expression by about 50-fold. The RDE also can be the 3′-UTR ofIFN-γ and the micro-RNA sites removed can be the MIR-125 sites.

The dynamic range of expression (control) with an RDE can be increasedby optimizing the codons of the transgene controlled by the RDE. Byincreasing the GC content of the wobble position of the codons in atransgene the efficiency of translation can be increased by 1-2 logs(10-100 fold). The increased efficiency of translation means the amountof expression in the “on” state with the RDE is increased. If the “off”state expression rate is not changed or changed less, the overalldynamic range of control with the RDE is increased.

New RDEs can be obtained from synthetic libraries made bycombinatorially mixing and matching parts of known RDEs by applyingtechniques such as molecular breeding and/or directed evolution to the3′-UTRs of genes known to have an RDE. For example, multiple 3′-UTRswith different RDEs are fragmented and assembled into synthetic 3′-UTRsthat are then screened or selected for RDE activity. RDEs with desiredproperties can be discovered from such libraries using positive and/ornegative selections.

Alternative Splicing

Alternative splicing can link a change in metabolic state in a cell tothe splicing of a pre-mRNA transcript into a mRNA that encodes and istranslated into a desired polypeptide. For example, following a changein metabolic state of a cell, a pre-mRNA transcript can undergoalternative splicing to produce a payload in the cell. Prior to thealternative splicing the pre-mRNA transcript can be spliced into atranscript encoding a nonsense polypeptide or into an mRNA encoding adifferent polypeptide product.

hnRNPLL (heterogeneous nuclear ribonucleoprotein L like) is a RNAbinding polypeptide that is made when immune cells (e.g., T-cells andB-cells) are activated (change in metabolic state). hnRNPLL is a masterregulator of activation-induced alternative splicing in lymphocytes,including T cells and B-cells. In T-cells, hnRNPLL effects the splicingof a variety of transcripts including CD45, a tyrosine phosphataseessential for T-cell development and activation.

hnRNPLL binds to CA repeats and also to C rich motifs, A rich motifs,and T rich motifs including, for example, CACACA(CA)_(n), CTTCCt/c,CATt/a, CATT, and TTTAt/aA. When hnRNPLL binding sites are in the3′-UTRs of a transcript, binding of hnRNPLL to the site can stabilizethe transcript. When hnRNPLL binding sites are within about 1 kilobaseon the 5′ side of an exon, hnRNPLL binding promotes inclusion of theexon during splicing. When hnRNPLL binding sites are within about 1kilobase on the 3′ side of an exon, hnRNPLL binding promotes exclusionof the exon. When hnRNPLL binds to a transcript it can alter thesplicing pattern of the transcript resulting in a new mRNA transcriptand new noncoding excision products (excised introns or introns plusexons). This alternative splicing pattern can produce an alternativelyspliced mRNA that now encodes a desired polypeptide (e.g., a payload),and/or the new noncoding excision products can encode a miRNA or siRNAthat is only produced upon alternative splicing.

hnRNPLL alternative splicing can be used alone or in combination withother regulatory signals such as RDEs. Together hnRNPLL alternativesplicing and RDEs can control expression of a payload upon activation ofa cell (change in metabolic state) when it reaches a target site havinga ligand that binds to a CAR, T-cell receptor (TCR), or other receptorexpressed on the cell. Binding of ligand to the receptor (e.g., CAR orTCR) activates the cell, increases expression of a payload by RDEmechanisms, and produces alternative splicing following hnRNPLLexpression. Together the RDE control and alternative splicing producedesired amounts of a payload or other transgene.

hnRNPLL alternative splicing can also be used to turn off a gene that isbeing expressed while the cell is quiescent. When the quiescent cell isactivated by ligand binding to a receptor (e.g., CAR or TCR) thischanges the metabolic state of the cell and hnRNPLL is expressed. If agene being expressed while the cell is quiescent has hnRNPLL bindingsites the binding of hnRNPLL can alter the splicing of the transcript sothat the mRNA no longer encodes the gene product. For example, a cell(e.g., T-cell or B-cell) can be engineered to express a stemness factor(e.g., Tox and/or TCF7) while the cell is quiescent. The nucleic acidencoding Tox and/or TCF7 can include intron(s) with hnRNPLL bindingsites so that in the absence of hnRNPLL the transcripts are spliced tomake the Tox and/or TCF7 polypeptides, but upon cell activation andexpression of hnRNPLL these transcripts now undergo alternative splicingand no longer produce transcripts encoding Tox and/or TCF7.

hnRNPLL alternative splicing also produces new “introns” or noncodingexcision products as well as new mRNAs. The new introns can encodeactive RNAs (e.g., miRNA, siRNA, etc.) that are only expressed in thenew introns. This adds yet another level of control to hnRNPLLalternative splicing as the miRNAs or siRNAs can turn off genes in theactivated cells that are detrimental to the effector state of the cellincluding, for example, Tox, SCF7, and other exhaustion factors. miRNAsand siRNAs that could be controlled in this manner include, for example,miR155, mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p, miR-491-5p,miR-541-3p, hsa-mir-26b-5p (MIRT030248), and hsa-mir-223-3p(MIRT054680).

Chimeric Antigen Receptors

Chimeric antigen receptors (CARs) can be fused proteins comprising anextracellular antigen-binding/recognition element, a transmembraneelement that anchors the receptor to the cell membrane and at least oneintracellular element. These CAR elements are known in the art, forexample as described in patent application US20140242701, which isincorporated by reference in its entirety for all purposes herein. TheCAR can be a recombinant polypeptide expressed from a constructcomprising at least an extracellular antigen binding element, atransmembrane element and an intracellular signaling element comprisinga functional signaling element derived from a stimulatory molecule. Thestimulatory molecule can be the zeta chain associated with the T cellreceptor complex. The cytoplasmic signaling element may further compriseone or more functional signaling elements derived from at least onecostimulatory molecule. The costimulatory molecule can be chosen from4-1BB (i.e., CD137), CD27 and/or CD28. The CAR may be a chimeric fusionprotein comprising an extracellular antigen recognition element, atransmembrane element and an intracellular signaling element comprisinga functional signaling element derived from a stimulatory molecule. TheCAR may comprise a chimeric fusion protein comprising an extracellularantigen recognition element, a transmembrane element and anintracellular signaling element comprising a functional signalingelement derived from a co-stimulatory molecule and a functionalsignaling element derived from a stimulatory molecule. The CAR may be achimeric fusion protein comprising an extracellular antigen recognitionelement, a transmembrane element and an intracellular signaling elementcomprising two functional signaling elements derived from one or moreco-stimulatory molecule(s) and a functional signaling element derivedfrom a stimulatory molecule. The CAR may comprise a chimeric fusionprotein comprising an extracellular antigen recognition element, atransmembrane element and an intracellular signaling element comprisingat least two functional signaling elements derived from one or moreco-stimulatory molecule(s) and a functional signaling element derivedfrom a stimulatory molecule. The CAR may comprise an optional leadersequence at the amino-terminus (N-term) of the CAR fusion protein. TheCAR may further comprise a leader sequence at the N-terminus of theextracellular antigen recognition element, wherein the leader sequenceis optionally cleaved from the antigen recognition element (e.g., ascFv) during cellular processing and localization of the CAR to thecellular membrane.

Chimeric Antigen Receptor—Extracellular Element

Exemplary extracellular elements useful in making CARs are described,for example, in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

The extracellular element(s) can be obtained from the repertoire ofantibodies obtained from the immune cells of a subject that has becomeimmune to a disease, such as for example, as described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes.

The extracellular element may be obtained from any of the wide varietyof extracellular elements or secreted proteins associated with ligandbinding and/or signal transduction as described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, U.S. Pat. Nos.5,359,046, 5,686,281 and 6,103,521, all of which are incorporated byreference in their entirety for all purposes.

The extracellular element can also be obtained from a variety ofscaffold protein families which share the common feature of a proteinscaffold core with protein loops that can confer binding specificity andwhich loops can be altered to provide different binding specificities.Knottins are one such scaffold protein that has peptide loops which canbe engineered to produce different binding specificities. For example,knottins can be engineered to have high affinity for specific integrinpeptides. See for example, Silverman et al., J. Mol. Biol. 385:1064-75(2009) and Kimura et al, Proteins 77:359-69 (2009), which areincorporated by reference in their entirety for all purposes. Somecancers overexpress certain integrin peptides and such cancers can betargeted by CARs that have an extracellular element that is a knottinspecific for the overexpressed integrin. One such integrin is theintegrin αvβ6 which is upregulated in multiple solid tumors such asthose derived from colon, lung, breast, cervix, ovary/fallopian tubes,pancreas, and head and neck. See for example, Whilding et al., Biochem.Soc. Trans. 44:349-355 (2016), which is incorporated by reference in itsentirety for all purposes.

The extracellular element can also be derived from knottins, which are afamily of peptides containing a disulfide bonded core that confersoutstanding proteolytic resistance and thermal stability. Knottins,which naturally function as protease inhibitors, antimicrobials, andtoxins, are composed of several loops that possess diverse sequencesamongst family members. Knottins can be engineered to include additionaldiversity of sequence in the loops to increase and create new bindingspecificities. Engineered knottins can be made to bind desired targets(e.g., desired antigens) with a desired specificity. Some Knottins bindwith nM specificity to integrins and can be used to target a CAR to acertain integrins (e.g., αvβ3/αvβ5, αvβ3/αvβ5/α5β1, or αvβ6 integrins).Integrins such as αvβ6 can be upregulated on solid tumors and so can besuitable targets for a CAR. Such αvβ6 integrin specific CARs can be madeusing a knottin specific for the αvβ6 integrin as the extracellularelement of the CAR. Activation of an engineered cell (e.g., a T-cell)through the αvβ6 knottin-CAR can be used to deliver a pay load to asolid tumor under the control of an RDE that causes expression of thepayload upon CAR cell activation.

In an aspect, the extracellular domain can be an antibody or otherbinding molecule that binds specifically to an onco-sialylated CD43 thatis widely found on AML and MDS blasts. See Hazenberg et al., EuropeanHematology Associate abstracts, Abst 5511 (2016), which is incorporatedby reference in its entirety for all purposes. The antibody AT14-013binds a specific sialylated epitope on the onco-sialylated CD43 whichepitope is not found on CD43 associated with normal cells and tissue.See WO 2016/209079 and WO 2015/093949, both of which are incorporated byreference in their entirety for all purposes. This antibody orantibodies or other binding molecules which compete for onco-sialylatedCD43 binding with AT14-013 are used to make anti-onco sialylated CD43CARs. For example, the variable regions of the heavy and light chain ofAT14-013 can be taken and reformatted as a single chain antibody for useas the extracellular domain of a CAR. Such an extracellular domain on aCAR directs the CAR cell (e.g., anti-onco sialylated CD43 CART-lymphocyte) to the AML and/or MDS cells targeting them for cellkilling or modification by the CAR cell.

Other tumor associated antigens that can be the target of the CARinclude, for example, c-Met (e.g., NSCLC), gpNMB (e.g., melanoma, breastcancer, other solid tumors), TRAP-2 (e.g., epithelial tumors and othersolid tumors), CEACAMS (e.g., colorectal cancer), CD56 (e.g., SCLC),CD25 (e.g., hematological cancers), guanyl cyclase C (e.g., pancreaticcancer), CAG (e.g., solid tumors), LIV-1 (e.g., breast cancer), PTK7(e.g., lung cancer, colorectal cancer, breast cancer, and ovariancancer), LAMP-1 (e.g., colorectal cancer, melanoma, laryngeal cancer),P-cadherin 3 (e.g., epithelial tumors), HER-3 (e.g., breast cancer),CD133 (e.g., hepatocellular carcinoma, pancreatic cancer, colorectalcancer, cholangiocarcinoma), BCMA (e.g., multiple myeloma), CD138 (e.g.,multiple myeloma), Ig kappa light chain (e.g., leukemia, lymphoma, NHL,and multiple myeloma), CD30 (e.g., NHL, HD), IL13Ra2 (e.g.,glioblastoma), and ligands for NKG2D (e.g., using the NKG2D receptor asthe binding domain for, e.g., AML, MDS, and MM).

Other tumor associated antigens that can be the target of the CARinclude, for example, mesothelin, disialoganglioside (GD2), Her-2, MUC1,GPC3, EGFRVIII, CEA, CD19, EGFR, PSMA, GPC2, folate receptor β, IgG Fcreceptor, PSCA, PD-L1, EPCAM, Lewis Y Antigen, L1CAM, FOLR, CD30, CD20,EPHA2, PD-1, C-MET, ROR1, CLDN18.2, NKG2D, CD133, TSHR, CD70, ERBB, AXL,Death Receptor 5, VEGFR-2, CD123, CD80, CD86, TSHR, ROR2, CD147, kappaIGG, IL-13, MUC16, IL-13R, NY-ESO-1, IL13RA2, DLL3, FAP, LMP1, TSHR,BCMA, NECTIN-4, MG7, AFP (alpha-fetoprotein), GP100, B7-H3, Nectin-4,MAGE-A1, MAGE-A4, MART-1, HBV, MAGE-A3, TAA, GP100, Thyroglobulin, EBV,HPV E6, PRAME, HERV-E, WT1, GRAS G12V, p53, TRAIL, MAGE-A10, HPV-E7,KRAS G12D, MAGE-A6, CD19, BCMA, CD22, CD123, CD20, CD30, CD33, CD138,CD38, CD7, SLAMF7, IGG FC, MUC1, Lewis Y Antigen, CD133, ROR1, FLT3,NKG2D, Kappa light chain, CD34, CLL-1, TSLP, CD10, PD-L1, CD44V6, EBV,CD5, GPC3, CD56, integrin B7, CD70, MUCL, CKIT, CLDN18.2, TRBC1, TAC1,CD56, and CD4.

Still other tumor associated antigens that can be the target of the CARinclude, for example, CD2, CD18, CD27, CD37, CD72, CD79A, CD79B, CD83,CD117, CD172, ERBB3, ERBB4, DR5, HER2, CS1, IL-1RAP, ITGB7, SLC2A14,SLC4A1, SLC6A11, SLC7A3, SLC13A5, SLC19A1, SLC22A12, SLC34A1, slc45A3,SLC46A2, Fra, IL-13Ra2, ULBP3, ULBP1, CLD18, NANOG, CEACAM8, TSPAN16,GLRB, DYRK4, SV2C, SIGLEC8, RBMXL3, HIST1HIT, CCR8, CCNB3, ALPPL2, ZP2,OTUB2, LILRA4, GRM2, PGG1, NBIF3, GYPA, ALPP, SPATA19, FCRLI, FCRLA,CACNG3, UPK3B, 12UMO4, MUC12, HEPACAM, BPI, ATP6V0A4, HMMR, UPK1A,ADGRV1, HERC5, C3AR1, FASLG, NGB, CELSR3, CD3G, CEACAM3, TNFRSFBC,MS4AB, S1PR5, EDNRB, SCN3A, ABCC8, ABCB1, ANO1, KCND2, HTR4, CACNB4,HTR4, CNR2, 26LRB, EXOC1, ENTPP1, ICAM3, ABCGB, SCN4B, SPN, CD68, ITGAL,ITGAM, SCTR, CYYR1, CLCN2, SLARA3, and JAG3.

Intracellular Element

The intracellular element can be a molecule that can transmit a signalinto a cell when the extracellular element of the Smart CAR (RNA controldevice—CAR), DE-CAR (destabilizing element—CAR), RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR(collectively “CARS”) binds to (interacts with) an antigen. Theintracellular signaling element can be generally responsible foractivation of at least one of the normal effector functions of theimmune cell in which the CAR(s) has been introduced. The term “effectorfunction” refers to a specialized function of a cell. Effector functionof a T cell, for example, may be cytolytic activity or helper activityincluding the secretion of cytokines. Thus the term “intracellularsignaling element” refers to the portion of a protein which transducesthe effector function signal and directs the cell to perform aspecialized function. While the entire intracellular signaling domaincan be employed, in many cases the intracellular element orintracellular signaling element need not consist of the entire domain.To the extent that a truncated portion of the intracellular signalingdomain is used, such truncated portion may be used as long as ittransduces the effector function signal. The term intracellularsignaling element is thus also meant to include any truncated portion ofthe intracellular signaling domain sufficient to transduce the effectorfunction signal. Examples of intracellular signaling elements for use inthe Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR of the invention include thecytoplasmic sequences of the T cell receptor (TCR) and co-receptors thatact in concert to initiate signal transduction following antigenreceptor engagement, as well as any derivative or variant of thesesequences and any recombinant sequence that has the same functionalcapability.

Intracellular elements and combinations of polypeptides useful with oras intracellular elements are described, for example, in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes.

Transmembrane Element and Spacer Element

The CAR, and/or RDE-CAR may comprise a transmembrane element. Thetransmembrane element can be attached to the extracellular element ofCAR, and/or RDE-CAR. The transmembrane element can include one or moreadditional amino acids adjacent to the transmembrane region, e.g., oneor more amino acid associated with the extracellular region of theprotein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/orone or more additional amino acids associated with the intracellularregion of the protein from which the transmembrane protein is derived(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of theintracellular region). The transmembrane element can be associated withone of the other elements used in the CAR, and/or RDE-CAR. Thetransmembrane element can be selected or modified by amino acidsubstitution to avoid binding of such elements to the transmembraneelements of the same or different surface membrane proteins, e.g., tominimize interactions with other members of the receptor complex. Thetransmembrane element can be modified to remove cryptic splice sites(e.g., CARS made with a CD8 transmembrane domain can be engineered toremove a cryptic splice site) and/or a transmembrane element can be usedin the CAR construct that does not have cryptic splice sites. Thetransmembrane element can be capable of homodimerization with anotherCAR, and/or RDE-CAR on the cell surface. The amino acid sequence of thetransmembrane element may be modified or substituted so as to minimizeinteractions with the binding elements of the native binding partnerpresent in the same cell.

Transmembrane elements useful in the present invention are described,for example, in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

Chimeric Antigen Receptors: Side-CARs

Side CARs, selection of Side CARs, and their use with or without atether are described, for example, in U.S. patent application Ser. No.15/070,352 filed on Mar. 15, 2016, and U.S. patent application Ser. No.15/369,132 filed Dec. 5, 2016, both of which are incorporated byreference in their entirety for all purposes.

Receptors

CARS may be used as the receptor with the cell and the RDE-transgene.CARS are described above. In addition to CARS, other receptors may beused to activate or otherwise change conditions in a cell so that atransgene under the control of an RDE is expressed. Receptors thatrecognize and respond to a chemical signal can be coupled to expressionof the transgene through the RDE. For example, ion channel-linked(ionotropic) receptors, G protein-linked (metabotropic) receptors, andenzyme-linked receptors can be coupled to the expression of thetransgene.

One class of receptor that can be coupled to transgene expression areimmune receptors such as, for example, T-cell receptors, B-cellreceptors (aka antigen receptor or immunoglobulin receptor), and innateimmunity receptors.

T-cell receptors are heterodimers of two different polypeptide chains.In humans, most T cells have a T-cell receptor made of an alpha (a)chain and a beta (β) chain have a T-cell receptor made of gamma anddelta (γ/δ) chains (encoded by TRG and TRD, respectively). Techniquesand primers for amplifying nucleic acids encoding the T-cell receptorchains from lymphocytes are well known in the art and are described in,for example, SMARTer Human TCR a/b Profiling Kits sold commercially byClontech, Boria et al., BMC Immunol. 9:50-58 (2008); Moonka et al., J.Immunol. Methods 169:41-51 (1994); Kim et al., PLoS ONE 7:e37338 (2012);Seitz et al., Proc. Natl Acad. Sci. 103:12057-62 (2006), all of whichare incorporated by reference in their entirety for all purposes. TheTCR repertoires can be used as separate chains to form an antigenbinding domain. The TCR repertoires can be converted to single chainantigen binding domains. Single chain TCRs can be made from nucleicacids encoding human alpha and beta chains using techniques well-knownin the art including, for example, those described in U.S. PatentApplication Publication No. US2012/0252742, Schodin et al., Mol.Immunol. 33:819-829 (1996); Aggen et al., “Engineering HumanSingle-Chain T Cell Receptors,” Ph.D. Thesis with the University ofIllinois at Urbana-Champaign (2010) a copy of which is found atideals.illinois.edu/bitstream/handle/2142/18585/AggenDavid.pdf?sequence=1, all of which are incorporated by reference intheir entirety for all purposes.

B-cell receptors include an immunoglobulin that is membrane bound, asignal transduction moiety, CD79, and an ITAM. Techniques and primersfor amplifying nucleic acids encoding human antibody light and heavychains are well-known in the art, and described in, for example,ProGen's Human IgG and IgM Library Primer Set, Catalog No. F2000;Andris-Widhopf et al., “Generation of Human Fab Antibody Libraries: PCRAmplification and Assembly of Light and Heavy Chain Coding Sequences,”Cold Spring Harb. Protoc. 2011; Lim et al., Nat. Biotechnol. 31:108-117(2010); Sun et al., World J. Microbiol. Biotechnol. 28:381-386 (2012);Coronella et al., Nucl. Acids. Res. 28:e85 (2000), all of which areincorporated by reference in their entirety for all purposes. Techniquesand primers for amplifying nucleic acids encoding mouse antibody lightand heavy chains are well-known in the art, and described in, forexample, U.S. Pat. No. 8,143,007; Wang et al., BMC Bioinform.7(Suppl):S9 (2006), both of which are incorporated by reference in theirentirety for all purposes. The antibody repertoires can be used asseparate chains in antigen binding domains, or converted to single chainantigen binding domains. Single chain antibodies can be made fromnucleic acids encoding human light and heavy chains using techniqueswell-known in the art including, for example, those described in Pansriet al., BMC Biotechnol. 9:6 (2009); Peraldi-Roux, Methods Molc. Biol.907:73-83 (2012), both of which are incorporated by reference in theirentirety for all purposes. Single chain antibodies can be made fromnucleic acids encoding mouse light and heavy chains using techniqueswell-known in the art including, for example, those described in Imai etal., Biol. Pharm. Bull. 29:1325-1330 (2006); Cheng et al., PLoS ONE6:e27406 (2011), both of which are incorporated by reference in theirentirety for all purposes.

Innate immunity receptors include, for example, the CD94/NKG2 receptorfamily (e.g., NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H), the 2B4receptor, the NKp30, NKp44, NKp46, and NKp80 receptors, the Toll-likereceptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, RP105).

G-protein linked receptors also known as seven-transmembrane domainreceptors are a large family of receptors that couple receptor bindingof ligand to cellular responses through G proteins. These G-proteins aretrimers of α, β, and γ subunits (known as Gα, Gβ, and Gγ, respectively)which are active when bound to GTP and inactive when bound to GDP. Whenthe receptor binds ligand it undergoes a conformational change andallosterically activates the G-protein to exchange GTP for bound GDP.After GTP binding the G-protein dissociates from the receptor to yield aGα-GTP monomer and a Gβγ dimer. G-protein linked receptors have beengrouped together into classes which include, for example, Rhodopsin-likereceptors, secretin receptors, metabotropic glutamate/pheromonereceptors, fungal mating pheromone receptors, cyclic AMP receptors, andfrizzled/smoothened receptors. G-protein receptors are used in a widevariety of physiological processes including detection ofelectromagnetic radiation, gustatory sense (taste), sense of smell,neurotransmission, immune system regulation, growth, cell densitysensing, etc.

Enzyme linked receptors also known as a catalytic receptor, is atransmembrane receptor, where the binding of an extracellular ligandcauses enzymatic activity on the intracellular side. Enzyme linkedreceptors have two domains joined together by a transmembrane portion(or domain) of the polypeptide. The two terminal domains are anextracellular ligand binding domain and an intracellular domain that hasa catalytic function. There are multiple families of enzyme linkedreceptors including, for example, the Erb receptor family, the glialcell-derived neurotrophic factor receptor family, the natriureticpeptide receptor family, the trk neurotrophin receptor family, and thetoll-like receptor family.

Ion channel linked receptors also known as ligand-gated ion channels arereceptors that allow ions such as, for example, Na⁺, K⁺, Ca²⁺ and Cl⁻ topass through the membrane in response to the binding of a ligand to thereceptor. There are multiple families of ligand-gated ion channelsincluding, for example, cationic cys-loop receptors, anionic cys-loopreceptors, ionotropic glutamate receptors (AMPA receptors, NMDAreceptors), GABA receptors, 5-HT receptors, ATP-gated channels, andPIP2-gated channels.

Eukaryotic Cells

Various eukaryotic cells can be used as the eukaryotic cell. Theeukaryotic cells can be animal cells. The eukaryotic cells can bemammalian cells, such as mouse, rat, rabbit, hamster, porcine, bovine,feline, or canine. The mammalian cells can be cells of primates,including but not limited to, monkeys, chimpanzees, gorillas, andhumans. The mammalians cells can be mouse cells, as mice routinelyfunction as a model for other mammals, most particularly for humans(see, e.g., Hanna, J. et al., Science 318:1920-23, 2007; Holtzman, D. M.et al., J Clin Invest. 103(6):R15-R21, 1999; Warren, R. S. et al., JClin Invest. 95: 1789-1797, 1995; each publication is incorporated byreference in its entirety for all purposes). Animal cells include, forexample, fibroblasts, epithelial cells (e.g., renal, mammary, prostate,lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, andhematopoietic cells. The animal cells can be adult cells (e.g.,terminally differentiated, dividing or non-dividing) or embryonic cells(e.g., blastocyst cells, etc.) or stem cells. The eukaryotic cell alsocan be a cell line derived from an animal or other source.

The eukaryotic cells can be stem cells. A variety of stem cells typesare known in the art and can be used as the eukaryotic cell, includingfor example, embryonic stem cells, inducible pluripotent stem cells,hematopoietic stem cells, neural stem cells, epidermal neural crest stemcells, mammary stem cells, intestinal stem cells, mesenchymal stemcells, olfactory adult stem cells, testicular cells, and progenitorcells (e.g., neural, angioblast, osteoblast, chondroblast, pancreatic,epidermal, etc.). The stem cells can be stem cell lines derived fromcells taken from a subject.

The eukaryotic cell can be a cell found in the circulatory system of amammal, including humans. Exemplary circulatory system cells include,among others, red blood cells, platelets, plasma cells, T-cells, naturalkiller cells, B-cells, macrophages, neutrophils, or the like, andprecursor cells of the same. As a group, these cells are defined to becirculating eukaryotic cells of the invention. The eukaryotic cell canbe derived from any of these circulating eukaryotic cells. Transgenesmay be used with any of these circulating cells or eukaryotic cellsderived from the circulating cells. The eukaryotic cell can be a T-cellor T-cell precursor or progenitor cell. The eukaryotic cell can be ahelper T-cell, a cytotoxic T-cell, a memory T-cell, a regulatory T-cell,a natural killer T-cell, a mucosal associated invariant T-cell, a gammadelta T cell, or a precursor or progenitor cell to the aforementioned.The eukaryotic cell can be a natural killer cell, or a precursor orprogenitor cell to the natural killer cell. The eukaryotic cell can be aB-cell, or a B-cell precursor or progenitor cell. The eukaryotic cellcan be a neutrophil or a neutrophil precursor or progenitor cell. Theeukaryotic cell can be a megakaryocyte or a precursor or progenitor cellto the megakaryocyte. The eukaryotic cell can be a macrophage or aprecursor or progenitor cell to a macrophage.

The eukaryotic cells can be obtained from a subject. The subject may beany living organisms. The cells can be derived from cells obtained froma subject. Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Anynumber of T cell lines available in the art also may be used. T-cellscan be obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficollseparation. Cells from the circulating blood of an individual can beobtained by apheresis. The apheresis product typically containslymphocytes, including T cells, monocytes, granulocytes, B cells, othernucleated white blood cells, red blood cells, and platelets. The cellscollected by apheresis may be washed to remove the plasma fraction andto place the cells in an appropriate buffer or media for subsequentprocessing steps. The cells can be washed with phosphate buffered saline(PBS). In an alternative aspect, the wash solution lacks calcium and maylack magnesium or may lack many if not all divalent cations. Initialactivation steps in the absence of calcium can lead to magnifiedactivation.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. Cells can be enrichedby cell sorting and/or selection via negative magnetic immunoadherenceor flow cytometry using a cocktail of monoclonal antibodies directed tocell surface markers present on the cells. For example, to enrich forCD4+ cells, a monoclonal antibody cocktail typically includes antibodiesto CD14, CD20, CD11b, CD16, HLA-DR, and CD8. It may be desirable toenrich for regulatory T cells which typically express CD4+, CD25+,CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, Tregulatory cells are depleted by anti-C25 conjugated beads or othersimilar method of selection.

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005, each of which is incorporated by reference in its entiretyfor all purposes.

NK cells may be expanded in the presence of a myeloid cell line that hasbeen genetically modified to express membrane bound IL-15 and 4-1BBligand (CD137L). A cell line modified in this way which does not haveWIC class I and II molecules is highly susceptible to NK cell lysis andactivates NK cells. For example, K562 myeloid cells can be transducedwith a chimeric protein construct consisting of human IL-15 maturepeptide fused to the signal peptide and transmembrane domain of humanCD8a and GFP. Transduced cells can then be single-cell cloned bylimiting dilution and a clone with the highest GFP expression andsurface IL-15 selected. This clone can then be transduced with humanCD137L, creating a K562-mb15-137L cell line. To preferentially expand NKcells, peripheral blood mononuclear cell cultures containing NK cellsare cultured with a K562-mb15-137L cell line in the presence of 10 IU/mLof IL-2 for a period of time sufficient to activate and enrich for apopulation of NK cells. This period can range from 2 to 20 days,preferably about 5 days. Expanded NK cells may then be transduced withthe anti-CD19-BB-ξ chimeric receptor.

Modification of CD3 Epsilon in T-lymphocytes

T-lymphocytes can be modified to reduce graft versus host reactions. Forexample, allogenic T-lymphocytes can be modified so that upontransplantation graft versus host reactions are reduced. The allogenicT-lymphocytes can be used in a CAR therapy and/or can carry othertransgenes for delivery by the T-lymphocyte at a target site. TheT-lymphocytes can be modified by introducing a mutant that has adominant effect on T-cell receptor function. For example, the mutant canknock out the T-cell receptor, or can disrupt signaling from the T-cellreceptor. Mutations of the CD3 epsilon chain can have such dominantnegative effects on T-cell receptor signaling. An example of a negativedominant mutant of CD3 epsilon is a C119S/C122S double mutant thatalters the C-X-X-C motif in the CD3 epsilon to S-X-X-S. This doublemutant is defective for signal transduction from an activated T-cellreceptor and so binding of host antigens by a T-lymphocyte (e.g., anallogenic T-lymphocyte) does not activate the T-lymphocyte reducinggraft versus host disease.

The CD3 epsilon double mutant (C119S/C122S) can be introduced into theT-lymphocyte by integrating it to the CD3 epsilon locus of theT-lymphocyte, or because this double mutant is a negative dominant itcan be introduced at other sites in the T-lymphocyte genome or can betransiently transfected/transduced into the T-lymphocyte. Transienttransfection can produce T-lymphocytes with CD3 epsilon double mutantassociated with the T-cell receptors of the T-lymphocytes prior toactivation by binding of a CAR (or other receptor) at the target site.If the transient expression has ended when the T-lymphocyte reachestarget and interacts with its CAR, the CAR T-lymphocytes can killthrough CAR reactions and through graft versus host/tumor cell reactionsthrough the allogenic T-cell receptors.

In an example, T-lymphocytes are obtained from a host (e.g., anallogenic host) and activated with CD3/CD28 beads. These activatedT-lymphocytes are transfected with a construct encoding a CAR (and/orother desired transgene) and a double mutant CD3 epsilon (C119S/C122S).T-lymphocytes transfected/transduced with the CAR are isolated (e.g., byaffinity isolation with the CAR or a selection against activeT-lymphocytes with active TCRs can be done) and administered to asubject.

Another mutation of CD3 epsilon can make deletions of one or more of theITAM portions of the polypeptide. These ITAM deletions reduce thesignaling capacity of CARS made with these ITAM mutants. The CD3 epsilonof the CAR can be engineered to have ITAM deletions of one or more ITAMsequences. T-cells engineered with a CAR and these CD3 epsilon geneswith the AITAM(s) can be used with payloads under control of an RDE.When these T-cells are activated by the CAR the payload is expressed.The AITAMs reduce the responsiveness of the cell to the CAR (a crippledCAR) which can reduce or prevent T-cell exhaustion. This allows theT-cell to produce the transgene payload for a longer period of time.

Nucleic Acids

Also described in this disclosure are nucleic acids that encode, atleast in part, the individual peptides, polypeptides, proteins, RDEs,and other post-transcriptional control devices described herein. Thenucleic acids may be natural, synthetic or a combination thereof. Thenucleic acids of the invention may be RNA, mRNA, DNA or cDNA.

The nucleic acids of the invention also include expression vectors, suchas plasmids, or viral vectors, or linear vectors, or vectors thatintegrate into chromosomal DNA. Expression vectors can contain a nucleicacid sequence that enables the vector to replicate in one or moreselected host cells. Such sequences are well known for a variety ofcells. The origin of replication from the plasmid pBR322 is suitable formost Gram-negative bacteria. In eukaryotic host cells, e.g., mammaliancells, the expression vector can be integrated into the host cellchromosome and then replicate with the host chromosome. Similarly,vectors can be integrated into the chromosome of prokaryotic cells.

Expression vectors also generally contain a selection gene, also termeda selectable marker. Selectable markers are well-known in the art forprokaryotic and eukaryotic cells, including host cells of the invention.Generally, the selection gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. An exemplary selection schemecan utilize a drug to arrest growth of a host cell. Those cells that aresuccessfully transformed with a heterologous gene produce a proteinconferring drug resistance and thus survive the selection regimen. Otherselectable markers for use in bacterial or eukaryotic (includingmammalian) systems are well-known in the art.

An example of a promoter that is capable of expressing a Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR transgene in a mammalian T cell is theEF1a promoter. The native EF1a promoter drives expression of the alphasubunit of the elongation factor-1 complex, which is responsible for theenzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1a promoterhas been extensively used in mammalian expression plasmids and has beenshown to be effective in driving CAR expression from transgenes clonedinto a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8):1453-1464 (2009), which is incorporated by reference in its entirety forall purposes. Another example of a promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Other constitutive promoter sequences may also be used, including, butnot limited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus promoter (MMTV), human immunodeficiency virus (HIV) longterminal repeat (LTR) promoter, MoMuLV promoter, phosphoglycerate kinase(PGK) promoter, MND promoter (a synthetic promoter that contains the U3region of a modified MoMuLV LTR with myeloproliferative sarcoma virusenhancer, see, e.g., Li et al., J. Neurosci. Methods vol. 189, pp. 56-64(2010) which is incorporated by reference in its entirety for allpurposes), an avian leukemia virus promoter, an Epstein-Barr virusimmediate early promoter, a Rous sarcoma virus promoter, as well ashuman gene promoters such as, but not limited to, the actin promoter,the myosin promoter, the elongation factor-1a promoter, the hemoglobinpromoter, and the creatine kinase promoter. Further, the invention isnot limited to the use of constitutive promoters.

Inducible or repressible promoters are also contemplated for use in thisdisclosure. Examples of inducible promoters include, but are not limitedto a Nuclear Factor of Activated T-cell inducible promoter (NFAT), ametallothionein promoter, a glucocorticoid promoter, a progesteronepromoter, a tetracycline promoter, a c-fos promoter, the T-REx system ofThermoFisher which places expression from the human cytomegalovirusimmediate-early promoter under the control of tetracycline operator(s),and RheoSwitch promoters of Intrexon. Macian et al., Oncogene20:2476-2489 (2001); Karzenowski, D. et al., BioTechiques 39:191-196(2005); Dai, X. et al., Protein Expr. Purif 42:236-245 (2005); Palli, S.R. et al., Eur. J. Biochem. 270:1308-1515 (2003); Dhadialla, T. S. etal., Annual Rev. Entomol. 43:545-569 (1998); Kumar, M. B, et al., J.Biol. Chem. 279:27211-27218 (2004); Verhaegent, M. et al., Annal. Chem.74:4378-4385 (2002); Katalam, A. K., et al., Molecular Therapy 13:S103(2006); and Karzenowski, D. et al., Molecular Therapy 13:S194 (2006),U.S. Pat. Nos. 8,895,306, 8,822,754, 8,748,125, 8,536,354, all of whichare incorporated by reference in their entirety for all purposes.Inducible promoter also include promoters with heat shock elements thatrespond to mild hyperthermia. Heat shock elements are made of multipleinverted repeats of the consensus sequence 5′-nGAAn-3′ located upstreamof a promoter such as, for example, a promoter from a heat shock gene(e.g., HSPB1).

Expression vectors typically have promoter elements, e.g., enhancers, toregulate the frequency of transcriptional initiation. Typically, theseare located in the region 30-110 bp upstream of the start site, althougha number of promoters have been shown to contain functional elementsdownstream of the start site as well. The spacing between promoterelements frequently is flexible, so that promoter function is preservedwhen elements are inverted or moved relative to one another. In thethymidine kinase (tk) promoter, the spacing between promoter elementscan be increased to 50 bp apart before activity begins to decline.Depending on the promoter, it appears that individual elements canfunction either cooperatively or independently to activatetranscription.

The expression vector may be a bi-cistronic construct or multiplecistronic construct. The two cistrons may be oriented in oppositedirections with the control regions for the cistrons located in betweenthe two cistrons. When the construct has more than two cistrons, thecistrons may be arranged in two groups with the two groups oriented inopposite directions for transcription. Exemplary bicistronic constructsare described in Amendola et al., Nat. Biotechnol. 23:108-116 (2005),which is incorporated by reference in its entirety for all purposes. Thecontrol region for one cistron may be capable of high transcriptionactivity and the other may have low transcriptional activity underconditions of use. One or both control regions may be inducible.Examples of high transcription activity control regions include, forexample, MND, EF1-alpha, PGK1, CMV, ubiquitin C, SV40 early promoter,tetracycline-responsive element promoter, cell-specific promoters, humanbeat-actin promoter, and CBG (chicken beta-globin), optionally includingthe CMV early enhancer. Examples of low transcription activity controlregions include, for example, TRE3G (commercially sold by Clontech, atetracycline-responsive element promoter with mutations that reducebasal expression), T-REx™ (commercially sold by ThermoFisher), and aminimal TATA promoter (Kiran et al., Plant Physiol. 142:364-376 (2006),which is incorporated by reference in its entirety for all purposes),HSP68, and a minimal CMV promoter. Examples of inducible control regionsinclude, for example, NFAT control regions (Macian et al, Oncogene20:2476-2489 (2001)), and the inducible control regions described above.

The bi-cistronic construct may encode a CAR and a polypeptide that is apayload (or makes a payload) to be delivered at a target site. Exemplarypayloads are described above and below. The nucleic acid encoding theCAR can be operably linked to a strong promoter, a weak promoter, and/oran inducible promoter, and optionally, operably linked to a RNA controldevice, DE, RDE, or combination of the foregoing. The CAR can be encodedby nucleic acids in a Side-CAR format. The nucleic acid encoding thepolypeptide can be operably linked to a strong promoter, a weakpromoter, and/or an inducible promoter. The nucleic acid encoding thepolypeptide that is a payload (or makes the payload) can be under thecontrol of an RDE. The RDE may be one that responds to the activationstate of the cell through, for example, glycolytic enzymes such as, forexample, glyceraldehyde phosphate dehydrogenase (GAPDH), enolase (ENO1or ENO3), phosphoglycerate kinase (PGK1), triose phosphate isomerase(TPI1), aldolase A (ALDOA), or phosphoglycerate mutase (PGAM1). The RDEmay also be bound and regulated by other energy metabolism enzymes suchas, for example, transketolase (TKT), malate dehydrogenase (MDH2),succinyl CoA Synthetase (SUGLG1), ATP citrate lyase (ACLY), orisocitrate dehydrogenase (IDH1/2). The host cell can express a CAR thatbinds to its antigen at a target site in a subject. This binding ofantigen at the target site activates the cell causing the cell toincrease glycolysis which induces expression of the nucleic acidencoding the polypeptide under the control of the RDE (bound byglycolytic or other energy metabolism enzymes).

The multicistronic constructs can have three or more cistrons with eachhaving control regions (optionally inducible) and RDEs operably linkedto some or all of the transgenes. These cassettes may be organized intotwo groups that are transcribed in opposite directions on the construct.Two or more transgenes can be transcribed from the same control regionand the two or more transgenes may have IRES (internal ribosome entrysite) sequences operably linked to the downstream transgenes.Alternatively, the two or more transgenes are operably linked togetherby 2A elements as described in Plasmids 101: Multicistronic Vectorsfound at blog.addgene.org/plasmids-101-multicistrnic-vectors. Commonlyused 2A sequences include, for example, EGRGSLLTCGDVEENPGP (T2A) (SEQ IDNO: 17), ATNFSLLKQAGDVEENPGP (P2A) (SEQ ID NO: 18); QCTNYALLKLAGDVESNPGP(E2A) (SEQ ID NO: 19); and VKQTLNFDLLKLAGDVESNPGP (F2A) (SEQ ID NO: 20)all of which can optionally include the sequence GSG at the aminoterminal end. This allows multiple transgenes to be transcribed onto asingle transcript that is regulated by a 3′-UTR with an RDE (or multipleRDEs).

The bicistronic/multicistronic vector can increase the overallexpression of the two or more cistrons (versus introducing the cistronson separate constructs). The bicistronic/multicistronic construct can bederived from a lenti-virus vector. The bicistronic/multicistronicconstruct can encode a CAR and a polypeptide(s) that is encoded on atransgene(s) (e.g., a payload), and the bicistronic construct mayincrease expression of the polypeptide encoded by the transgene(s) whenthe cell is activated by the CAR.

Expression constructs can be modified to remove an RNA splice site in a5′-LTR of the construct so as to increase the transduction frequency ofthe expression construct. Many lentiviral transduction constructs have aresidual splice site in the 5′-LTR that can reduce transductionfrequency through splicing events with this site that alter the nucleicacids to be introduced. For example, lentiviral vectors represented bythe pCDH vectors (System Biosciences), the lentiviral vectors pLVTH,pRRLSIN, pWPI, and pWPXL, as well as other lentiviral vectors contain aportion of the HIV 5′-LTR that includes a splice acceptor site. Removalof this splice site from the vectors increases transduction frequencieswith the modified vectors (compared to the non-modified vectors). Thesplice donor site in the residual HIV 5′-LTR segment can be disrupted bymaking two nucleotide changes, G290C and U291A (numbering the HIV splicesite according to Keane et al., Science 348:917-921 (2015), which isincorporated by reference in its entirety for all purposes). Otherchanges in this splice donor site could also be made to knock out thissplice site. Other transduction vectors also include residual splicesites that can disrupt the desired sequence aftertransduction/transfection and removal of these splice sites shouldincrease transduction/transfection frequencies with these vectors.

It may be desirable to modify polypeptides described herein. One ofskill will recognize many ways of generating alterations in a givennucleic acid construct to generate variant polypeptides Such well-knownmethods include site-directed mutagenesis, PCR amplification usingdegenerate oligonucleotides, exposure of cells containing the nucleicacid to mutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques (see,e.g., Gillam and Smith, Gene 8:81-97, 1979; Roberts et al., Nature328:731-734, 1987, which is incorporated by reference in its entiretyfor all purposes). The recombinant nucleic acids encoding thepolypeptides of the invention can be modified to provide preferredcodons which enhance translation of the nucleic acid in a selectedorganism.

The polynucleotides can also include polynucleotides includingnucleotide sequences that are substantially equivalent to otherpolynucleotides described herein. Polynucleotides can have at leastabout 80%, more typically at least about 90%, and even more typically atleast about 95%, sequence identity to another polynucleotide. Thenucleic acids also provide the complement of the polynucleotidesincluding a nucleotide sequence that has at least about 80%, moretypically at least about 90%, and even more typically at least about95%, sequence identity to a polynucleotide encoding a polypeptiderecited herein. The polynucleotide can be DNA (genomic, cDNA, amplified,or synthetic) or RNA. Methods and algorithms for obtaining suchpolynucleotides are well known to those of skill in the art and caninclude, for example, methods for determining hybridization conditionswhich can routinely isolate polynucleotides of the desired sequenceidentities.

Nucleic acids which encode protein analogs or variants (i.e., whereinone or more amino acids are designed to differ from the wild typepolypeptide) may be produced using site directed mutagenesis or PCRamplification in which the primer(s) have the desired point mutations.For a detailed description of suitable mutagenesis techniques, seeSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and/or CurrentProtocols in Molecular Biology, Ausubel et al., eds, Green PublishersInc. and Wiley and Sons, N.Y (1994), each of which is incorporated byreference in its entirety for all purposes. Chemical synthesis usingmethods well known in the art, such as that described by Engels et al.,Angew Chem Intl Ed. 28:716-34, 1989 (which is incorporated by referencein its entirety for all purposes), may also be used to prepare suchnucleic acids.

Amino acid “substitutions” for creating variants are preferably theresult of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, i.e., conservative aminoacid replacements. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Also disclosed herein are nucleic acids encoding a transgene, includinga transgene encoding a CARS. The nucleic acid encoding the transgene canbe easily prepared from an amino acid sequence of the specified CARcombined with the sequence of the RNA control device by a conventionalmethod. A base sequence encoding an amino acid sequence can be obtainedfrom the aforementioned NCBI RefSeq IDs or accession numbers of GenBenkfor an amino acid sequence of each element, and the nucleic acid of thepresent invention can be prepared using a standard molecular biologicaland/or chemical procedure. For example, based on the base sequence, anucleic acid can be synthesized, and the nucleic acid of the presentinvention can be prepared by combining DNA fragments which are obtainedfrom a cDNA library using a polymerase chain reaction (PCR).

The nucleic acids can be linked to another nucleic acid so as to beexpressed under control of a suitable promoter. The nucleic acid can bealso linked to, in order to attain efficient transcription of thenucleic acid, other regulatory elements that cooperate with a promoteror a transcription initiation site, for example, a nucleic acidcomprising an enhancer sequence, a polyA site, or a terminator sequence.In addition to the nucleic acid of the present invention, a gene thatcan be a marker for confirming expression of the nucleic acid (e.g. adrug resistance gene, a gene encoding a reporter enzyme, or a geneencoding a fluorescent protein) may be incorporated.

When the nucleic acid is introduced into a cell ex vivo, the nucleicacid of may be combined with a substance that promotes transference of anucleic acid into a cell, for example, a reagent for introducing anucleic acid such as a liposome or a cationic lipid, in addition to theaforementioned excipients. Alternatively, a vector carrying the nucleicacid of the present invention is also useful. Particularly, acomposition in a form suitable for administration to a living body whichcontains the nucleic acid of the present invention carried by a suitablevector is suitable for in vivo gene therapy.

Introducing Nucleic Acids into Eukaryotic Cells

A process for producing a cell expressing a transgene includes a step ofintroducing the nucleic acid encoding the transgene described hereininto a eukaryotic cell. This step can be carried out ex vivo. Exemplarymethods for introducing nucleic acids to eukaryotic cells are described,for example, in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

Virus Payloads

Viruses can be used to deliver transgenes to target cells. Viruses cancarry nucleic acid constructs (e.g., transfer plasmids) as payloads andso deliver to a target cell desired nucleic acids for modification ofthe target cell genotype and/or phenotype (transiently or stably). Inmany of these transduction applications, the nucleic acid carried by thevirus does not include all of the viral genome, and often includes theviral genome signals needed for packaging the nucleic acid constructinto the virus without most or all of the rest of the viral genome. Forexample, lentiviral helper plasmid and transfer plasmids systems fortransduction of target cells are available from addgene. Other helperand transfer plasmid systems are commercially available form a number ofsources (e.g., Clontech/Takara).

When used as a payload, synthesis of viral capsids, packaging of payloadnucleic acids, and release of virus with payload nucleic acids can berestricted to the target site by timing the expression of the virusgenes for replication and coat proteins to binding of ligand by areceptor at the target site. Such control can be achieved using RDEsthat induce expression when the cell undergoes a change in metabolicstate (e.g., activation of glycolysis after receptor binding to target).This RDE control can regulate expression of master switch factors forexpression of the virus genes. For example, a transcription regulatoryfactor can be placed under the control of a suitable RDE, and the viralgenes for replication, coat proteins etc can be placed under the controlof this transcription factor. When the host cell binds to ligand at thetarget site through an appropriate receptor (e.g., a CAR) this activatesthe cell, induces expression of the transcription factor with theappropriate RDE leading to expression of the viral replication proteins,coat proteins, etc.

Transduction with a Viral Vector

Transduction can be accomplished with a virus vector such as aretrovirus vector (including an oncoretrovirus vector, a lentivirusvector, and a pseudo type vector), an adenovirus vector, anadeno-associated virus (AAV) vector, a simian virus vector, a vacciniavirus vector or a sendai virus vector, an Epstein-Barr virus (EBV)vector, and a HSV vector can be used. The virus vector can lackreplicating ability so as not to self-replicate in an infected cell.

When a retrovirus vector is used to transduce the host cell, the processcan be carried out by selecting a suitable packaging cell based on a LTRsequence and a packaging signal sequence possessed by the vector andpreparing a retrovirus particle using the packaging cell. Examples ofthe packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078),GP+E-86 and GP+envAm-12 (U.S. Pat. No. 5,278,056, which is incorporatedby reference in its entirety for all purposes), and Psi-Crip(Proceedings of the National Academy of Sciences of the United States ofAmerica, vol. 85, pp. 6460-6464 (1988), which is incorporated byreference in its entirety for all purposes). A retrovirus particle canalso be prepared using a 293 cell or a T cell having high transfectionefficiency. Many kinds of retrovirus vectors produced based onretroviruses and packaging cells that can be used for packaging of theretrovirus vectors are widely commercially available from manycompanies.

A number of viral based systems have been developed for gene transferinto mammalian cells. A selected gene can be inserted into a vector andpackaged in viral particles using techniques known in the art. Therecombinant virus can then be isolated and delivered to cells of thesubject either in vivo or ex vivo. A number of viral systems are knownin the art. Adenovirus vectors can be used. A number of adenovirusvectors are known in the art and can be used. In addition, lentivirusvectors can be used.

A viral vector derived from a RNA virus can be used to introduce to acell a RDE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/ortransgene-RDE encoding polynucleotides. The RNA virus vector can encodethe reverse complement or antisense strand of the polynucleotideencoding the RNA control device and CAR construct (the complementarystrand encodes the sense strand for the RNA control device, DE, RDE, CARand/or Side-CAR construct). Thus, the RNA control device should not beactive in the single stranded, RNA virus vector. The sense strand of theRNA virus construct encoding the RNA control device, DE, RDE, CAR,Side-CAR, and/or transgene can be used, and the viral vector with theRNA control device, DE, RDE, CAR and/or Side-CAR construct is maintainedand replicated in the presence (or absence) of ligand for the sensorelement of the RNA control device (or under conditions where the RDE isstable) to prevent cleavage of the RNA. The viral vector encoding thesense strand of the RNA control device, DE, RDE, CAR, Side-CAR, and/ortransgene construct in the viral vector can then be maintained andreplicated with (or without) ligand for the sensor element. Transductionefficiency can be increased by cryopreservation of packaged constructsprior to thawing and transduction. Increased efficiency ranged fromabout 10% to about 70%.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise a CARSand/or transgene-RDE expressing cell, e.g., a plurality of CARS and/ortransgene-RDE expressing cells, as described herein, in combination withone or more pharmaceutically or physiologically acceptable carriers,diluents or excipients. Such compositions may comprise buffers such asneutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arein one aspect formulated for intravenous administration.

Pharmaceutical compositions may be administered in a manner appropriateto the disease to be treated (or prevented). The quantity and frequencyof administration will be determined by such factors as the condition ofthe patient, and the type and severity of the patient's disease,although appropriate dosages may be determined by clinical trials.

Suitable pharmaceutically acceptable excipients are well known to aperson skilled in the art. Examples of the pharmaceutically acceptableexcipients include phosphate buffered saline (e.g. 0.01 M phosphate,0.138 M NaCl, 0.0027 M KCl, pH 7.4), an aqueous solution containing amineral acid salt such as a hydrochloride, a hydrobromide, a phosphate,or a sulfate, saline, a solution of glycol or ethanol, and a salt of anorganic acid such as an acetate, a propionate, a malonate or a benzoate.An adjuvant such as a wetting agent or an emulsifier, and a pH bufferingagent can also be used. The pharmaceutically acceptable excipientsdescribed in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.1991) (which is incorporated herein by reference in its entirety for allpurposes) can be appropriately used. The composition can be formulatedinto a known form suitable for parenteral administration, for example,injection or infusion. The composition may comprise formulationadditives such as a suspending agent, a preservative, a stabilizerand/or a dispersant, and a preservation agent for extending a validityterm during storage.

A composition comprising the eukaryotic cells described herein as anactive ingredient can be administered for treatment of, for example, acancer (blood cancer (leukemia), solid tumor (ovarian cancer) etc.), aninflammatory disease/autoimmune disease (pemphigus vulgaris, lupuserythematosus, rheumatoid arthritis, asthma, eczema), hepatitis, and aninfectious disease the cause of which is a virus such as influenza andHIV, a bacterium, or a fungus, for example, a disease such astuberculosis, MRSA, VRE, or deep mycosis, depending on an antigen towhich a CAR, DE-CAR, and/or Side-CAR polypeptide binds.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, intranasally, intraarterially,intratumorally, into an afferent lymph vessel, by intravenous (i.v.)injection, or intraperitoneally. In one aspect, the T cell compositionsof the present invention are administered to a patient by intradermal orsubcutaneous injection. In one aspect, the T-cell compositions of thepresent invention are administered by i.v. injection. The compositionsof T-cells may be injected directly into a tumor, lymph node, or site ofinfection. The administration can be done by adoptive transfer.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). A pharmaceutical composition comprising the eukaryotic cellsdescribed herein may be administered at a dosage of 10⁴ to 10⁹ cells/kgbody weight, in some instances 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. A eukaryotic cellcomposition may also be administered multiple times at these dosages.Eukaryotic cells can also be administered by using infusion techniquesthat are commonly known in immunotherapy (see, e.g., Rosenberg et al.,New Eng. J. of Med. 319:1676, 1988, which is incorporated by referencein its entirety for all purposes).

Uses of Eukaryotic Cells

Nucleic acids encoding CARS and/or transgene-RDE(s) can be used toexpress CAR, DE-CAR, Side-CAR, and/or transgene polypeptides ineukaryotic cells. The eukaryotic cell can be a mammalian cell, includingfor example human cells or murine cells. The eukaryotic cells may alsobe, for example, hematopoietic cells including, e.g., T-cells, naturalkiller cells, B-cells, or macrophages.

T-cells (e.g., CD4+ or CD8+) or natural killer cells can be engineeredwith a polynucleotide encoding a CAR. Ligand for the RNA control device,DE, or Side CAR is added to the T-cells (e.g., CD4+ or CD8+) or naturalkiller cells can be added in increasing amounts to obtain a desiredamount of effector function. The desired amount of effector function canbe an optimized amount of effector function with a known amount (and/ordensity) of target antigen on target cells. Effector function can betarget cell killing, activation of host immune cells, cytokinesecretion, production of granzymes, production of apoptosis inducingligands, production of other ligands that modulate the immune system,etc. The effector function can be secretion of cytokines such as, forexample, IL-2, IFN-γ, TNF-α, TGF-β, and/or IL-10. Effector function canbe the killing of target cells. Target cells can be killed withgranzymes. Target cells can be induced to undergo apoptosis. Eukaryoticcells with CARs can kill target cells through apoptosis and granzymes.

The RDE, DE, RNA control device, or Side CAR regulatory element can beused to control expression of a transgene. This transgene expression candeliver a payload at a target site. These transgenes can also be carriedby viral constructs, or viruses when the payload is a virus. Expressionof the transgene can cause a desired change in the eukaryotic cell. AnRDE regulated by GAPDH can be used for payload delivery, and theeukaryotic cell (e.g., T-cell, natural killer cell, B-cell, macrophage,dendritic cell, or other antigen presenting cell) can be activated(e.g., by a CAR) when it reaches the target site. Upon activation of theeukaryotic cell at the target site through the CAR, the cell inducesglycolysis and the GAPDH releases from the RDE allowed payloadexpression and delivery. The target site can be a tumor or infection andthe transgene could encode a cytokine, a chemokine, an antibody, acheckpoint inhibitor, a granzyme, an apoptosis inducer, complement, anenzyme for making a cytotoxic small molecule, an enzyme that cleavespeptides or saccharides (e.g., for digesting a biofilm), other cytotoxiccompounds, or other polypeptides that can have a desired effect at thetarget site. Checkpoint inhibitors include agents that act at immunecheckpoints including, for example, cytotoxic T-lymphocyte-associatedantigen 4 (CTLA4), programmed cell death protein (PD-1), Killer-cellImmunoglobulin-like Receptors (KIR), and Lymphocyte Activation Gene-3(LAG3). Examples of checkpoint inhibitors that may be used as payloadsinclude, for example, Nivolumab (Opdivo®), Pembrolizumab (Keytruda®),Cemiplimab (Libtayo®), Atezolizumab (Tecentriq®), Avelumab (Bavencio®),Durvalumab (Imfinzi®), Ipilimumab (Yervoy®), Lirilumab, and BMS-986016.Nivolumab, Atezolizumab and Pembrolizumab act at the checkpoint proteinPD-1 and inhibit apoptosis of anti-tumor immune cells. Some checkpointinhibitors prevent the interaction between PD-1 and its ligand PD-L1.Ipilimumab acts at CTLA4 and prevents CTLA4 from downregulatingactivated T-cells in the tumor. Lirilumab acts at KIR and facilitatesactivation of Natural Killer cells. BMS-986016 acts at LAG3 andactivates antigen-specific T-lymphocytes and enhances cytotoxic Tcell-mediated lysis of tumor cells.

The payload can be one or more of an anti-IL33 antibody, anti-LAG3antibody, anti-TIM3 antibody, anti-TIGIT antibody, anti-MARCO antibody,anti-VISTA antibody, anti-CD39 antibody, anti-41BB antibody, IL-15,IL-21, IL-12, CD40L, and/or Leptin. The IL-33 receptor is upregulated inT_(regs) (regulatory T-cells) and anti-IL33 antibody reducesproliferation and activation of T_(regs). Anti-LAG3 antibody can alsodecrease activity of T_(regs). Anti-Il33 antibody and anti-LAG3 antibodycan be used alone or together to reduce the activity of T_(regs) whichcan reduce the suppression of CAR T-cells and other anti-cancer T-cells.Anti-TIM-3 antibody allows co-localization of CD8+ T-cells and DC-1cells (which improves anti-tumor response). MARCO is expressed onmacrophages and in the tumor microenvironment this can be suppressive toT-cells. Anti-MARCO antibody prevents this tumor suppression bymacrophages. Anti-VISTA antibody reduces the amount of neutrophils inthe tumor microenvironment. A high neutrophil to T-cell ratio in thetumor microenvironment correlates with poor patient outcomes. Decreasingthe neutrophils in the tumor can improve patient outcomes and tumor cellkilling. IL-15 and Il-21 increase the expansion of natural killer cellsand Il-15 can rescue CD8+ T-cells and may prevent T-cell exhaustion.CD40L plays a central role in priming, co-stimulation and activation ofT-cells in an immune response. Anti-CD39 antibody can reduce adenosinelevels in the tumor microenvironment. High levels of adenosine in thetumor microenvironment can induce immunosuppression. Anti-CD39 antibodycan reduce this immunosuppression. Anti-41BB antibody can preventT-cells from undergoing apoptosis and can also cause tumor cells toupregulate expression of PD1 (so can be combined with anti-PD1therapies).

Cytokines can include, for example, IL-2, IL-12, IL-15, IL-18, IL-21,IFN-γ, TNF-α, TGF-β, and/or IL-10. Cytotoxic agents can include, forexample, granzymes, apoptosis inducers, complement, or a cytotoxic smallmolecule. The payload can be a gene regulatory RNA, such as, forexample, siRNA, microRNAs (e.g., miR155), shRNA, antisense RNA,ribozymes, and the like, or guide RNAs for use with CRISPR systems. Thepayload can be an anti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4antibody, anti-IL1b antibody,anti-IL33 antibody, anti-LAG3 antibody,anti-TIM3 antibody, anti-TIGIT antibody, anti-MARCO antibody, anti-VISTAantibody, anti-CD39 antibody, PGC-alpha, Leptin, a BiTE, CCL2,anti-CXCR4 antibody, anti-CXCL12 antibody, HAC, heparinase,hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA (e.g.,mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, Ox40-41BB, miRNAfor Tox (e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)),miRNA for TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p,miR-342-5p, miR-491-5p, miR-541-3p), anti-CD28 antibody (including fulllength and fragments such as single chain antibodies), IL-21, Leptin,GOT2, NAMPT, CD56, IL-2 superkine, anti-REGNASE-1 payloads (e.g.,miRNA), C-jun, cysteinase enzyme, cystinase enzyme, PCBP1 (poly(RC)binding protein 1), complement (one or more of B, C1-C9, D, C5b, C3b,C4b, C2a), BMP-1, anti-TGFb agents (e.g., anti-TGFb antibody, solubleTGFbR, anti-avB6 integrin antibody, natural TGFb binding proteins, smallmolecules such as GW788388, Tranilast, Losartan, HMG CoA reductaseinhibitors, Imatinib mesylate, PPAR-g agonists, rosiglitazone,Pirfenidone, Halofuginone), IL7 combined with CCL19 (e.g.,IL7-t2A-CCL19), dnTNFR2, dnTGFBR2, DCN, DKK1, OKT3, NOS2, CCL5,anti-4-1BB agonist Antibody, anti-CD11b, anti-CD28 agonist Ab,anti-CD29/anti-VEGF, anti-CTLA4 Ab, anti-IL1b Ab, BiTE, CCR2,CCR4/CXCL12 disruption, HAC, heparinase, HSP60, HSP70, hyaluronidase,IL-12, IL15, IL18, IL2, anti-CSF1R and anti-IGF1, anti-IL4, IL4 receptorantagonists, IL4 binders (e.g., soluble IL4R), soluble CD40 ligand(e.g., secreted ecto-CD40L), membrane CD40 ligand, a TGFBR antagonist,and/or 4-1BB ligand. The payloads can also include those found inUS20190183932, which is incorporated by reference in its entirety forall purposes. The payload delivered at a target site (e.g., non-tumortarget site) can be a factor that protects the target site such as, forexample, an anti-inflammatory, a factor that attracts T-regulatory cellsto the site, or cytokines or other factors that cause suppression andreduction in immune activity. The payload can be an enzyme that cleavespeptides or saccharides, for example hyaluronidase, heparanase,metalloproteinases and other proteinases which can be used, for example,to digest an undesired biofilm. Myeloid modifying payloads (“MMpayloads”) which reduce immune suppression or inhibition caused bymyeloid cells may be delivered including, for example, ApoE3, ApoE4,Hsp60, Hsp70, TNFα, antagonists of CSF1 receptor, CD40L (CD154) and/orIL-12. Two or more MM payloads can also be delivered by the CAR, DE-CAR,side-CAR and/or other receptor cell (e.g., T-cell) using RDEs thatproduce different pharmacokinetics for delivery. For example, thedifferent MM payloads could be controlled by different RDEs so that theCmax of delivery for the different MM payloads occurs at differenttimes. For example, Myeloid modifying payloads can promote activated M1macrophages that are proinflammatory and tumoricidal. A MM payload thatpromotes M1 phenotypes are antagonists of CSF1R (antagonists that blockand do not activate the CSF1 receptor and agents that bind CSF1 andprevent it from interacting with the CSF1R). Such antagonists of CSF1Rinclude, for example, small-molecule inhibitors, PLX3397 (Pexidartinib,Plexxikon), PLX7486 (Plexxikon), ARRY-332 (Array Biopharma),JNJ-40346527 (Johnson & Johnson), and BLZ945 (Novartis). Exemplaryantibodies which are antagonists of CSF include, for example,Emactuzumab (Roche), AMG820 (Amgen), IMC-CS4 (LY3022855, Eli Lilly), andMCS110 (Novartis). Cannarile et al, J. Immunotherp. Cancer 5:53 (2017)which is incorporated by reference in its entirety for all purposes. Thepayload can be localized to the target cell (e.g., tumor site) by fusingor associating the payload with a Small Leucine Rich Proteoglycans(SLRPs) such as Decorin, Biglycan, or fibromodulaon/Lumican. TheDecorin, Biglycan, or Lumican can bind to the collagen near the targetcell and this binding will localize the payload at or near the targetsite. This strategy is particularly useful for keeping cytotoxicpayloads localized to the target cells (e.g., a tumor). Decorin andBiglycan can also bind to TGF-beta at or near the target site and reducesuppression of the engineered T-cell, and so these can be used as apayload themselves to reduce TGFb. A Decorin, Biglycan, and/or lumicanpayload can also be constitutively expressed, or expressed under thecontrol of an RDE with a moderate level of baseline expression(mimicking low level constitutive expression coupled with increasedexpression upon cell activation). The payload can be one or more of anyof the above. The payload can be an imaging agent that allows the targetsite to be imaged. The payload may be a polypeptide that can be imageddirectly, or it can be a polypeptide that interacts with a substrate tomake a product that can be imaged, imaging polypeptides include, forexample, thymidine kinase (PET), dopamine D2 (D2R) receptor, sodiumiodide transporter (NIS), dexoycytidine kinase, somatostatin receptorsubtype 2, norepinephrine transporter (NET), cannabinoid receptor,glucose transporter (Glut1), tyrosinase, sodium iodide transporter,dopamine D2 (D2R) receptor, modified haloalkane dehalogenase,tyrosinase, β-galactosidase, and somatostatin receptor 2. These reporterpayloads can be imaged using, for example, optical imaging, ultrasoundimaging, computed tomography imaging, optical coherence tomographyimaging, radiography imaging, nuclear medical imaging, positron emissiontomography imaging, tomography imaging, photo acoustic tomographyimaging, x-ray imaging, thermal imaging, fluoroscopy imaging,bioluminescent imaging, and fluorescent imaging. These imaging methodsinclude Positron Emission Tomography (PET) or Single Photon EmissionComputed Tomography (SPECT).

AB toxins can be used to engineer fusion payloads that deliver a desiredprotein product into a target cell. AB toxins include, for examplediphtheria toxin, tetanus toxin, exotoxin A of P. aeruginosa, iota toxinIa of C. perfringens, C2 toxin CI of C. botulinum,ADP-ribosyltransferase of C. difficile, etc. The AB toxin can beengineered to replace the catalytic (toxic) component (A domain) of theAB toxin with the desired protein so that the modified AB toxin bindsits receptor and delivers the desired protein into a target cell throughthe B domain of the AB toxin. The B domain of AB toxins generallyincludes a receptor binding portion that binds to a feature on thetarget cell, and a transmembrane domain portion that forms a pore andinteracts with target cell proteins to transport the A domain into thetarget cell. The B toxin fusions replace the A domain with a desiredprotein that the B domain will transport into the target cell throughthe B domain pore utilizing the cytosolic translocation factor complex(CTF) from the target cell.

These B toxin fusions can also be engineered to replace the receptorbinding portion of the B domain with a binding domain (e.g., ligand)that binds to a desired antigen and/or receptor on a desired targetcell. These modified B toxin fusions now bind to a desired target celland then transport the desired protein into the target cell using the Bdomain pore and the CTF of the target cell. For example, the B toxinfusion could be engineered to bind to the antigen target of a CAR sothat the B toxin fusion delivers payload from a CAR cell into a targetcell (e.g., a tumor cell). The transgene payload can encode a cellpenetrating peptide. The cell penetrating peptide can be fused toanother protein to make a chimeric protein (aka fusion protein) in whichthe CPP portion guides the chimeric protein to the cytoplasm of a targetcell.

Multiple systems are envisioned for use that can kill target cellsdirectly. These include, for example, the introduction of a viral or abacterial gene into target cells. This approach turns a non-toxicpro-drug to a toxic one. There are systems that have been extensivelyinvestigated: the cytosine deaminase gene (“CD”) of Escherichia coli,which converts the pro-drug 5-Fluorocytosine (“5-FC”) to 5-Fluorouracil(“5-FU”); and the herpes simplex virus thymidine kinase gene (“HSV-tk”),which converts ganciclovir (“GCV”) to ganciclovir monophosphate,converted by the cancer cells' enzymes to ganciclovir triphosphate. TheHSV-tk/GCV system useful in killing tumor cells directly, involvesadenoviral transfer of HSV-tk to tumor cells, with the subsequentadministration of ganciclovir. Specifically, recombinantreplication-defective adenovirus is employed to transfer the thymidine,HSV-tk, into hepatocellular carcinoma (“HCC”) cells to confersensitivity to ganciclovir. Three useful HCC cell lines include, forexample, Hep3B, PLC/PRF/5 and HepG2, which can efficiently infect, invitro, by a recombinant adenovirus carrying lacZ reporter gene(“Ad-CMVlacZ”). Expression of HSV-tk in HCC cells infected with arecombinant adenovirus carrying HSV-tk gene (“AdCMVtk”) inducessensitivity to ganciclovir in a dose-dependent manner (Qian et al.,Induction of sensitivity to ganciclovir in human hepatocellularcarcinoma cells by adenovirus-mediated gene transfer of herpes simplexvirus thymidine kinase, Hepatology, 22:118-123 (1995))doi.org/10.1002/hep.1840220119.

When the payload is a gene regulatory RNA, such as, for example, siRNAs,shRNAs, and/or microRNAs (e.g., miR155), the regulatory RNA (e.g.,mir155) can be the transgene or can be included in an intron of atransgene encoding a polypeptide. For example, a mir155 cassette asdescribed in Du et al., FEBs J. 273:5421-27 (2006) and Chung et al.,Nucl Acids Res. 34:e53 (2006) can be used as the payload or beengineered into an intron of a transgene that is used as the payload.The mir155 cassette (or cassette for other regulatory RNA) can beengineered into a transgene as an intron or the transgene can be themir155 cassette, optionally with additional nucleotides. The regulatoryRNA transgene (or transgene with regulatory RNA as an intron) can beplaced under the control of an RDE. RDEs can impact RNA processing andstability in the nucleus. After the transgene encoding the regulatoryRNA (e.g., mir155) or encoding a transgene with a regulatory RNA (e.g.,mir155) intron is transcribed, the transcript can be processed in thenucleus by the nuclear microprocessor complex or other nuclearcomponents to make the nucleotide stem-loop precursor regulatory RNA(e.g., pre-mir155). The pre-regulatory RNA (e.g., pre-mir155) stem-loopis exported out of the nucleus where it is processed by Dicer to form ashort RNA duplex. The short RNA duplex(es) are bound by Argonaute (Ago)to form the core of the multi-subunit complex called the RNA-inducedsilencing complex (RISC). By operably linking a RDE to the transgeneencoding the regulatory RNA (e.g., mir155) or the transgene with theregulatory RNA (e.g., mir155) intron, the expression of regulatory RNA(e.g., mir155) can be regulated by the RDE. Different RDEs can beoperably linked to the regulatory RNA (e.g., mir155) transgene ortransgene with regulatory RNA (e.g., mir155) intron to provide differenttiming and kinetics of expression following activation of a eukaryoticcell (e.g., activation of a T-cell by the TCR or a CAR). RDEs can beused that produce expression quickly after activation of the cell (e.g.,AU2 or AU101), produce high expression 72-96 hours after activation(e.g., AU5 or AU21), or produce increasing expression through 192 hoursafter expression (e.g., AU19 or AU22). RDEs can also be selected thatwill produce continuous expression of regulatory RNA (e.g., mir155) orthat will produce expression for a period of time after activation ofthe cell followed by reduced expression. Multiple regulatory RNA (e.g.,mir155) constructs (e.g., with mir155 as the transgene or a transgenewith a mir155 intron) with different RDEs can be used to providecontinuous expression of regulatory RNA (e.g., mir155) followingactivation of a cell (e.g., T-cell) by using RDEs that provide differentpharmacokinetic profiles of expression which together produce continuousexpression (e.g., see Example 11). Alternatively, select RDEs orcombinations of RDEs or combinations of regulatory RNA (e.g., mir155)with different RDEs can be used to provide a desired expression profileof the regulatory RNA (e.g., mir155).

Upregulation of mir155 has been associated with activated CD8+ T-cellsand the formation of memory T-cells after an immunological challenge.Upregulation of mir155 expression during activation of T-cells (e.g.,CAR T-cells activated by target antigens) will potentiate the CAR T-cellresponse against target cells. Placing mir155 under control of aheterologous RDE (e.g., an RDE that responds to GAPDH) ties upregulationof mir155 to activation of the T-cell so that mir155 is upregulated inactivated T-cells (e.g., CD8+, CAR T-cells). This upregulation canincrease proliferation of activated T-cells. The upregulation can alsodecrease T-cell exhaustion and senescence. The upregulation can alsopotentiate T-cell effector functions resulting in increased target cellkilling.

Effector function of T-cells can also be enhanced by downregulating TCF7and/or Tox expression and/or by upregulating IL-15 expression. TCF7 is amember of the T-cell factor/lymphoid enhancer-binding factor family ofhigh mobility group (HMG) box transcriptional activators. This gene isexpressed predominantly in T-cells and plays a critical role in naturalkiller cell and innate lymphoid cell development. HMG box protein TCF7can be a regulator in the switch between self-renewal anddifferentiation. TCF7 can have a dual role in promoting the expressionof genes characteristic of self-renewing CD34+ cells while repressinggenes activated in partially differentiated CD34− state. TCF7 canregulate a network of transcription factors that switch cells from anaïve, undifferentiated state to a differentiated, effector cell state.When TCF7 is expressed cells adopt a self-renewal state that is morenaïve and less differentiated. TCF7 can be downregulated using miRNAssuch as, for example, mIR-192, mIR-34a, miR-133a, miR-138-5p,miR-342-5p, miR-491-5p, and/or miR-541-3p. In an example, one of more ofthese miRNAs can be encoded in one or more introns of a payload that arespliced out when the transcript is bound by hnRNPLL (see above), andwhen the payload is expressed in an activated cell making hnRNPLL thesemiRNAs will downregulate TCF7. Alternatively, a transgene encoding TCF7can be used as an off switch for activated CAR T-cells. If TCF7 isexpressed after the effector, CAR T-cell has killed the target cancercells, this should push the CAR T-cell into a naïve, undifferentiatedstate (an off state for the CAR T-cell). The transgene encoding TCF7could be placed under the control of an inducible promoter (e.g., aninducible promoter that is ligand inducible) or it could be placed undercontrol of an RDE that results in expression after eight days or more ofcell activation (e.g., see Example 11). Expression of TCF7 can be turnedoff by removal of ligand (or other inducing factors for the induciblepromoter), and/or the RDE control will turn off expression. This canreturn the CAR T-cell to state where it can be reactivated by binding totarget ligand at other cancer cells.

Thymocyte selection-associated high mobility group box (TOX) protein isa member of a small subfamily of proteins (TOX2, TOX3, and TOX4) thatshare almost identical HMG-box sequences. TOX can be induced by highantigen stimulation of the T cell receptor and TOX can be a centralregulator of T_(EX) (exhausted T-cells). Robust TOX expression canresult in commitment to development of the T_(EX) cell type. TOXexhaustion may counteract and balance T-cell overstimulation andactivation-induced cell death in settings of chronic antigenstimulation. Effector T-cells (e.g., activated CD8+ T-cells) can havelow Tox, whereas higher levels of Tox pushes the effector cells tobecome T_(EX) cells. TEX cells have reduced effector function but arestill effective against low level infections or small numbers of cancercells.

Effector function of T-cells can be enhanced by including a payloadencoding an miRNA for Tox (e.g., hsa-mir-26b-5p (MIRT030248)hsa-mir-223-3p (MIRT054680)) under regulation of an RDE. Followingactivation of the T-cell, the RDE control will result in expression ofthe miRNA for Tox. This miRNA will lower levels of Tox in the T-cellinhibiting T_(EX) formation by the activated T-cells resulting in moreactive, effector T-cells against a target. In addition, a payload can beTox itself, used as on off-switch that pushes the activated T-cells intoa T_(EX) phenotype at a desired time. When used as an off-switch, Toxexpression can be under control of an inducible promoter that can beinduced to express Tox at a desired time (e.g., by adding an appropriateligand), Tox can be controlled by an RNA control device or a DE (ligandcan induce expression), or Tox can be placed under control of an RDEthat produces expression at late time intervals after activation of thecell (e.g., see Example 11). Functional state and type of T-cell cantailored by treating T-cells with electromagnetic radiation.Electromagnetic radiation in the UV range can condition T-cells tobecome Treg cells. For example, a dose of UVA/UVB can induce formationof Tregs. Electromagnetic radiation in the blue light range can activateT-cells.

An exemplary payload is a transgene encoding ApoE (e.g., ApoE2, ApoE3and/or APoE4) which is secreted from the cell. ApoE can bind toreceptors (e.g., LRP8) on Myeloid Derived Suppressor Cells (MDSC) andreduce the survival of MDSCs. MDSCs are a heterogeneous population ofsuppressive innate immune cells that can expand in certain diseasestates. In some cancers (e.g., melanoma, lung, breast and ovariancancers) MDSC levels can substantially rise in the tumor(s) and in theplasma of patients. Such patients with high levels of circulating MDSCscan respond poorly to checkpoint blockade. MDSCs can mediateimmunosuppression in these patients and induce angiogenesis. Payloadexpression of ApoE (e.g., ApoE4) can reduce the number of MDSCs intumors and circulating in the serum, and result in suppression of tumorprogression and metastatic colonization. The reduction in MDSCs in thetumor also enables other immune cells (e.g., CAR T-cells) to moreefficiently kill tumor cells. The ApoE payload can also act directly onmyeloid malignancies that express the LRP8 receptor. In such examples,the payload delivery of ApoE to a myeloid cancer cell can suppressand/or kill the cancer cell. Thus, ApoE can be a payload for delivery tomyeloid malignancies that are LRP8+, including LRP8+ AML. Delivery ofthe ApoE payload by a eukaryotic cell (e.g., primary T-cell) can becombined with another therapeutic agent such as, for example, ananti-cancer agent (e.g., a CAR T-cell, a chemotherapeutic, radiation, acheckpoint inhibitor, or any of the anti-cancer therapeutics describedherein). The ApoE effect on MDSCs can potentiate the action of the otheranti-cancer agent.

Another exemplary payload is a transgene encoding NO-synthase (e.g.,iNOS, nNOS and eNOS). NO synthase can bind to GAPDH and can sequesterthe GAPDH allowing RDE (which are bound by GAPDH) controlled transgenes(or native genes) to be expressed, or increasing expression from RDE(which are bound by GAPDH, e.g., AU 19 (TMEM-219), AU 20 (TMEM-219snp),AU 21 (CCR7), AU 22 (SEM-A4D), and AU 23 (CDC42-SE2)) controlledtransgenes (or native genes) once the cell is activated and glycolysisis induced. Expression of NO synthase can induce RDE (which are bound byGAPDH) controlled expression (through binding to GAPDH) and/or canpotentiate RDE (which are bound by GAPDH) controlled expression bydecreasing the amount of GAPDH that can bind RDEs and/or increasing thetime over which GAPDH cannot bind to RDEs. When NO synthase is used toincrease the RDE (which are bound by GAPDH) response to cell activation,a transgene encoding NO synthase can be placed under control of an RDEso that when the cell is activated, expression from the transgeneencoding the NO synthase is induced. When NO synthase is used to induceexpression from RDE (which are bound by GAPDH) controlled genes, the NOsynthase can be placed under inducible control (e.g., induciblepromoters, RNA control devices, or destabilizing elements as disclosedin U.S. Pat. No. 9,777,064, which is hereby incorporated by reference inits entirety for all purposes) and induction of NO synthase expressioninduces expression from the RDE (which are bound by GAPDH) controlledgenes.

An exemplary payload is a transgene encoding HSV-Thymidine Kinase(HSV-TK). HSV-TK can be used as an adjuvant, and/or as a super antigenthat induces an inflammatory response in the patient. When used in thismanner, a cell secretes the HSV-TK payload at the target site inducingan inflammatory response. The transgene encoding the HSV-TK can also beused as a kill switch to eliminate the engineered cells (e.g., CART-cells with or without a RDE controlled payload). When used as a killswitch, the HSV-TK can be controlled by a late expressing RDE so theHSV-TK is expressed after the CAR T-cell has acted at the target site,or the transgene expressing the HSV-TK can be controlled by a ligandinducible control means so that the HSV-TK protein is expressed inresponse to the ligand which is introduced at a desired time. In thekill-switch application, ganciclovir can be provided to the cells andthe HSV-TK converts the ganciclovir to GCV-triphosphate which kills thecell by a cytotoxic effect. A transgene expressing HSV-TK can also beincluded in a viral payload so that when the virus infects target cellsthe target cells express HSV-TK. Ganciclovir is provided to the targetcells which use the HSV-TK to convert the ganciclovir toGCV-triphosphate which is toxic to the target cells.

Thymidine kinase can be used with PET reporter probes such as, forexample, [¹⁸F]9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)-guanine, afluorine-18-labelled penciclovir analogue, which when phosphorylated bythymidine kinase (TK) becomes retained intracellularly, or is 5-(76)Br-bromo-2′-fluoro-2′-deoxyuridine. The relevant reporter probes foreach of the PET reporters are well known to the skilled artisan. Anexemplary reporter probe for dopamine D2 (D2R) receptor is3-(2′-[¹⁸F]fluoroethyl)spiperone (FESP) (MacLaren et al., Gene Ther.6(5):785-91 (1999)). An exemplary reporter probe for the sodium iodidetransporter is ¹²⁴I, which is retained in cells following transport bythe transporter. An exemplary reporter probe for deoxycytidine kinase is2′-deoxy-2′-¹⁸F-5-ethyl-1-β-d-arabinofuranosyluracil (¹⁸F-FEAU). Anexemplary reporter probe for somatostatin receptor subtype 2 is^(99m/94m)Tc-, ⁹⁰Y-, or ¹⁷⁷Lu-labeled octreotide analogues, for example⁹⁰Y-, or ¹⁷⁷Lu-labeled DOTATOC (Zhang et al., J Nucl Med. 50(suppl2):323 (2009)); ⁶⁸Ga-DOTATATE; and ¹¹¹In-DOTABASS (see. e.g., Brader etal., J Nucl Med. 54(2):167-172 (2013), incorporated herein byreference). An exemplary reporter probe for norepinephrine transporteris ¹¹C-m-hydroxyephedrine (Buursma et al., J Nucl Med. 46:2068-2075(2005)). An exemplary reporter probe for the cannabinoid receptor is¹¹C-labeled CB2 ligand, ¹¹C-GW405833 (Vandeputte et al., J Nucl Med.52(7):1102-1109 (2011)). An exemplary reporter probe for the glucosetransporter is [¹⁸F]fluoro-2-deoxy-d-glucose (Herschman, H. R., Crit RevOncology/Hematology 51:191-204 (2004)). An exemplary reporter probe fortyrosinase is N-(2-(diethylamino)ethyl)-¹⁸F-5-fluoropicolinamide (Qin etal., Sci Rep. 3:1490 (2013)). Other reporter probes are described in theart, for example, in Yaghoubi et al., Theranostics 2(4):374-391 (2012),incorporated herein by reference.

An exemplary photoacoustic reporter probe for β-galactosidase is5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (Li et al., J BiomedOpt. 12(2):020504 (2007)). Exemplary X-ray reporter includes, amongothers, somatostatin receptor 2, or other types of receptor basedbinding agents. The reporter probe can have a radiopaque label moietythat is bound to the reporter probe and imaged, for example, by X-ray orcomputer tomography. Exemplary radiopaque label is iodine, particularlya polyiodinated chemical group (see, e.g., U.S. Pat. No. 5,141,739), andparamagnetic labels (e.g., gadolinium), which can be attached to thereporter probe by conventional means. Optical imaging agents include,for example, a fluorescent polypeptide. Fluorescent polypeptidesinclude, for example, green fluorescent protein from Aequorea victoriaor Renilla reniformis, and active variants thereof (e.g., bluefluorescent protein, yellow fluorescent protein, cyan fluorescentprotein, etc.); fluorescent proteins from Hydroid jellyfishes, Copepod,Ctenophora, Anthrozoas, and Entacmaea quadricolor, and active variantsthereof; and phycobiliproteins and active variants thereof. The opticalimaging agent can also be a bioluminescent polypeptide. These include,for example, aequorin (and other Ca⁺² regulated photoproteins),luciferase based on luciferin substrate, luciferase based onCoelenterazine substrate (e.g., Renilla, Gaussia, and Metridina), andluciferase from Cypridina, and active variants thereof.

CARS and/or universal-CARs can be designed to include receptors againstantigens that are of bacterial, fungal or viral origin. Because CARS canbe utilized to fight infections, which are a source of toxicity inimmunocompromised patients, such anti-pathogen CARS can be used inconjunction with CARS T-cell therapy specific for a TAA.

A eukaryotic cell can bind to a specific antigen via the CAR, DE-CAR,and/or Side-CAR polypeptide causing the CAR, DE-CAR, and/or Side-CARpolypeptide to transmit a signal into the eukaryotic cell, and as aresult, the eukaryotic cell can be activated and so express anappropriate RDE-transgene. The activation of the eukaryotic cellexpressing the CARS is varied depending on the kind of a eukaryotic celland the intracellular element of the CARS. The eukaryotic cell canexpress a RDE transcript that poises the cell for effector function uponstimulation of the eukaryotic cell through a CARS.

A eukaryotic cell expressing the RDE-transgene or RDE transcript, andoptionally, a CARS, T-cell receptor, B-cell receptor, innate immunityreceptor and/or other receptor or targeting polypeptide can be used as atherapeutic agent to treat a disease. The therapeutic agent can comprisethe eukaryotic cell expressing the RDE-transgene or RDE transcript, andoptionally, a CARS, T-cell receptor, B-cell receptor, innate immunityreceptor and/or other receptor or targeting polypeptide as an activeingredient, and may further comprise a suitable excipient. Examples ofthe excipient include pharmaceutically acceptable excipients for thecomposition. The disease against which the eukaryotic cell expressingthe RDE-transgene or RDE transcript, and optionally, a CARS, T-cellreceptor, B-cell receptor, innate immunity receptor and/or otherreceptor or targeting polypeptide is administered is not particularlylimited as long as the disease shows sensitivity to the eukaryotic celland/or the product of the RDE-transgene.

Examples of diseases that can be treated include a cancer (blood cancer(leukemia), solid tumor (ovarian cancer) etc.), an inflammatorydisease/autoimmune disease (asthma, eczema), hepatitis, and aninfectious disease, the cause of which is a virus such as influenza andHIV, a bacterium, or a fungus, for example, tuberculosis, MRSA, VRE, anddeep mycosis, other immune mediated diseases such as neurodegenerativediseases like Alzheimer's or Parkinson's, and metabolic diseases likediabetes. A receptor (e.g., a CAR) can target the eukaryotic cell to thediseased cell(s) and when the receptor binds to its target at thediseased cell(s) the receptor can send a signal into the eukaryotic cellleading to expression of the RDE-transgene. The RDE-transgene encodes apolypeptide that is useful in treating or killing the diseased cell(s).A cancer and/or solid tumor can be treated with a eukaryotic cellexpressing receptor that binds to a tumor associated (or cancerassociated) antigen, such as those described above. When the receptorbinds to the tumor associated antigen the receptor sends a signal intothe cell that causes the RDE-transgene to be expressed (e.g., the signaleffects an RDE binding protein leading to expression of theRDE-transcript). The RDE-transcript can encode a polypeptide thatactivates the eukaryotic cell so that the eukaryotic cell treats thecancer and/or the RDE-transcript encodes a polypeptide that itselftreats the cancer (e.g., a cytotoxic polypeptide).

An autoimmune disease (e.g., pemphigus vulgaris, lupus erythematosus,rheumatoid arthritis, multiple sclerosis, Crohn's disease) can betreated with a eukaryotic cell expressing a RDE-transgene or RDEtranscript, and optionally, a CARS, T-cell receptor, B-cell receptor,innate immunity receptor and/or other receptor or targeting polypeptidethat binds to the immune proteins associated with the autoimmunedisease. The receptor or targeting polypeptide can trigger expression ofthe RDE-transgene that encodes a polypeptide useful in treating theautoimmune disease (e.g., the polypeptide can regulate the cells causingthe autoimmune disease or kill these cells). The eukaryotic cellexpressing the RDE-transgene or RDE transcript, and receptor ortargeting polypeptide can target cells that make an antibody involvedwith the autoimmune disease (e.g., the RDE-transgene can encode apolypeptide that kills the antibody producing cells or that inhibits theproduction of antibody by these cells). The eukaryotic cell expressingthe RDE-transgene or RDE transcript, and receptor or targetingpolypeptide can target T-lymphocytes involved with the autoimmunedisease (e.g., the RDE-transgene can encode a polypeptide that kills thetarget T-lymphocytes or that regulates the activity of theT-lymphocytes).

Eukaryotic cells expressing the RDE-transgene or RDE transcript, andoptionally, a CARS, T-cell receptor, B-cell receptor, innate immunityreceptor and/or other receptor or targeting polypeptide can be used as atherapeutic agent to treat an allergy. Examples of allergies that can betreated include, for example, allergies to pollen, animal dander,peanuts, other nuts, milk products, gluten, eggs, seafood, shellfish,and soy. The eukaryotic cell expressing the RDE-transgene or RDEtranscript, and receptor or targeting polypeptide can target cells thatmake an antibody which causes the allergic reaction against, forexample, pollen, animal dander, peanuts, other nuts, milk products,gluten, eggs, seafood, shellfish, and soy. The targeted cells can be oneor more of B-cells, memory B-cells, plasma cells, pre-B-cells, andprogenitor B-cells. Targeted cells can also include T-lymphocytes whichcause the allergic reaction against, for example, pollen, animal dander,peanuts, other nuts, milk products, gluten, eggs, seafood, shellfish,and soy. Eukaryotic cells expressing the RDE-transgene or RDEtranscript, and receptor or targeting polypeptide can bind to theidiotypic determinant of the antibody or T-cell receptor.

The eukaryotic cell expressing the RDE-transgene or RDE transcript, andoptionally, a CARS, T-cell receptor, B-cell receptor, innate immunityreceptor and/or other receptor or targeting polypeptide can beadministered for treatment of a disease or condition. For example, theeukaryotic cell can be utilized to treat an infectious disease. Theeukaryotic cell can express a receptor or targeting polypeptide thatbinds to an antigen found on the infectious disease causing agent or acell infected with such an agent. The receptor or targeting polypeptidebinds the antigen associated with the infectious disease and sends asignal into the eukaryotic cell that leads to expression of theRDE-transgene. The RDE-transgene encodes a product that can activate theeukaryotic cell for treating the infectious disease (e.g., theeukaryotic cell can produce a cytotoxic polypeptide or a cytokine thatactivates immune cells). The RDE-transgene can also encode a polypeptidethat itself is a cytotoxic polypeptide or a cytokine. The eukaryoticcell can also be utilized for prevention of an infectious disease (usedprophylactically), for example, after bone marrow transplantation orexposure to radiation, donor lymphocyte transfusion for the purpose ofremission of recurrent leukemia, and the like.

The therapeutic agent comprising the eukaryotic cell expressing theCARS, T-cell receptor, B-cell receptor, innate immunity receptor and/orother receptor or targeting polypeptide as an active ingredient can beadministered intradermally, intramuscularly, subcutaneously,intraperitoneally, intranasally, intraarterially, intravenously,intratumorally, or into an afferent lymph vessel, by parenteraladministration, for example, by injection or infusion, although theadministration route is not limited.

The RDE-transgene or RDE transcript, and optionally, CARS, T-cellreceptor, B-cell receptor, innate immunity receptor and/or otherreceptor or targeting polypeptide can be used with a T-lymphocyte thathas aggressive anti-tumor properties, such as those described in Pegramet al, CD28z CARs and armored CARs, 2014, Cancer J. 20(2):127-133, whichis incorporated by reference in its entirety for all purposes. The RDEtranscript can encode a polypeptide that causes aggressive anti-tumorproperties in the T-lymphocyte.

A transgene, a CAR, DE-CAR, and/or Side CAR polypeptides can becontrolled by an RDE from the 3′-UTR of the gene encoding IL-2 or the3′-UTR of IFN-γ. These RDEs can be modified to inactivate microRNA sitesfound in the RDE. Using these control elements makes expression of theCAR, DE-CAR, Side-CAR, and/or transgene sensitive to changes in theglycolytic state of the host cell through the interaction of the RDEwith glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When the hostcell is in a quiescent state a large proportion of the GAPDH is notinvolved in glycolysis and is able to bind to the RDE resulting inreduced translation of the transcript encoding the CAR, DE-CAR,Side-CAR, and/or transgene polypeptides. When the host cell is inducedto increase glycolysis, e.g., by providing the host cells with glucose,or other small molecules that will increase glycolytic activity, GAPDHbecomes enzymatically active and is not able to bind to the RDE. Thereduction in GAPDH binding to the RDE increases translation of thetranscripts (e.g., by increasing half-life of the transcript and/or byincreasing the translation rate) encoding the CAR, DE-CAR, Side-CAR, orother transgene. The glycolytic activity of GAPDH can be increased byincreasing the amount and/or activity of triose isomerase. The host cellcan be induced to over-express a recombinant triose isomerase, and thisover-expression increases the glycolytic activity of GAPDH. A glycolysisinhibitor can be added to decrease expression of the transcript with theRDE. Such glycolysis inhibitors include for example, dimethylfumarate(DMF), rapamycin, 2-deoxyglucose, 3-bromophyruvic acid, iodoacetate,fluoride, oxamate, ploglitazone, dichloroacetic acid, quinones (e.g.,chloroquine, hydroxychloroquine, etc.), or other metabolism inhibitorssuch as, for example, dehydroepiandrosterone. Expression from the RDEcontrolled transcript can be increased by the addition of GAPDH (orother RDE binding protein) inhibitor that inhibits binding of the RDE byGAPDH (or other RDE binding protein). Such GAPDH inhibitors include, forexample, CGP 3466B maleate or Heptelidic acid (both sold by Santa CruzBiotechnology, Inc.), pentalenolactone, or 3-bromopyruvic acid. Quinonessuch as, for example, chloroquine and hydroxychloroquine, can de-acidifythe endosome impairing antigen processing by APCs, decrease signalingfrom toll-like receptors, reduces T-cell proliferation, T-cell metabolicactivity, T-cell cytokine secretion, interferes with IL-2 production,and interferes with T-cell response to IL-2.

Constructs encoding transcripts with RDEs can be expressed in eukaryoticcells to bind to RDE binding proteins and so reduce the ability of thoseRDE binding proteins to interact with native transcripts in the cell.The recombinant transcripts can compete for binding of RDE bindingproteins and this can reduce the inhibition and/or activation of nativetranscripts within the eukaryotic cell by the RDE binding proteins. Theconstructs encoding transcripts with the RDEs can be used in this way tochange when and how native transcripts are expressed in the eukaryoticcell. The eukaryotic cell can be a T-cell, natural killer cell, orB-cell and the recombinant transcript has RDEs that are shared withcytokine or cytotoxic transcripts (e.g., in their 3′ untranslatedregions). The recombinant transcript can compete for binding with theRDE binding proteins (e.g., GAPDH and/or other glycolytic enzymesdescribed above) that regulate expression of the cytokine or cytotoxicpolypeptide and change the threshold (e.g., glycolysis activity forGAPDH) needed to express the cytokine or cytotoxic polypeptide. This canbe used to create super T-cell (aka Angry T-cells or Hornet T-cells)that will secrete higher amounts of cytokines and/or cytotoxic proteins(greater C_(max)) in response to stimulation of the immune cell (e.g.,through a CAR or T-cell receptor). T-cells can be reprogrammed with arecombinant transcript encoding an RDE from an IL-2 transcript so thatwhen the T-cell is stimulated by its T-cell receptor it produces moreIL-2 and other effector polypeptides with faster kinetics. Thesereprogrammed T-cells can also produce other inflammatory cytokines andcytotoxic polypeptides (e.g., granzymes and/or perforins) in largeramounts and with faster kinetics. Reprogramming T-cells and naturalkiller cells into such Angry/Hornet states can be useful for treatingdisease and disorders, including, for example, tumors, other cancers,and infectious diseases.

RDEs can be used to reduce CAR expression in immune cells until thoseimmune cells are activated by target or at a desired time. This canresult in expression of the CAR at desired times for therapeutic effectwhile reducing the systemic exposure of a subject to the CAR. Thereduced systemic exposure can reduce and/or inhibit the development ofan immune response against the CAR as the subject's immune system willsee less CAR over time.

Control of receptor (e.g., CAR and/or TCR) expression can be used tomodulate the PK-PD axis of an immunotherapy. The amount of receptorexpressed on the surface of cell can be modulated with the strength of apromoter, the inducibility of the promoter, the use of bicistronicconstructs with different promoter strengths expressing the twocistrons, RDEs (selection of RDE impacts dynamic range and timing ofexpression), GC3 content of the transcript, RNA control devices, degronsand/or Side-CARs. These control elements used singly or in combinationchange the amount of receptor on the surface of the cell which changesthe input signal (e.g., amount of ligand for the receptor) needed toactivate the cell so that it produces an output (e.g., payload deliveryor target cell killing). Using this control, the input signal needed forthe receptor cells can be optimized for a given target, compartment ofthe body, reduction of side effects, etc. as desired. RDEs can also beused to change the timing of the output from the cell after activationat the receptor (e.g., CAR and/or TCR).

Some neural degenerative diseases and syndromes are associated withinflammation, as are a number of other non-neural diseases andsyndromes. Such inflammation associated diseases can be treated, atleast in part, by providing a subject with small molecules (or othermolecules) that increase the availability of inhibitory RDE bindingproteins within immune cells. Such small molecules include, for example,glycolysis inhibitors (e.g., dimethylfumarate (DMF), rapamycin,2-deoxyglucose, 3-bromophyruvic acid, iodoacetate, fluoride, oxamate,ploglitazone, dichloroacetic acid), other metabolic inhibitors (e.g.,dehydroepiandrosterone), etc. For example, glycolytic inhibitors reduceglycolysis in the cell and can increase the amount of free GAPDH (notinvolved in glycolysis) for binding to RDEs reducing the expression ofthese transcripts. A number of inflammatory gene products in immunecells (e.g., gene products that activate the immune system) areregulated by RDEs that can bind GAPDH. Decreasing glycolysis increasesthe amount of free GAPDH for RDE binding, increases the amount of GAPDHbound to the RDEs of these inflammatory genes and reduces the expressionof these inflammatory genes. Inflammatory genes include proinflammatorycytokines such as, for example, IL-1, TNF-α, INF-g, and GM-CSF. Thesecytokines have 3′-UTRs with RDEs that can bind RDE binding proteins,including GAPDH, to regulate their expression. The increased GAPDH canbind to these RDEs and decrease the expression of these proinflammatorycytokines. Reduced expression of proinflammatory cytokines could reduceactivity of the immune system in these subjects reducing inflammation.The reduction in inflammation can have positive therapeutic effectsalleviating symptoms and/or treating the underlying disease state inthese inflammation related neural diseases, as well as in otherinflammation associated diseases and syndromes.

RDEs (e.g., AU elements) can be selected to provide maximal expressionat a desired time point and to provide a desired amount of polypeptideat that time point. RDEs can also be selected to provide a desired areaunder the curve for a polypeptide. As shown in Table 2 of Example 20,different RDEs (e.g., AU elements) reached maximal rates of expressionat different times. Also as shown in Table 1, different RDEs provideddifferent amounts of expression with different profiles over timeproviding different AUC. Using these RDEs in combination with differenttransgenes allows temporal programming of when the different transgenesreach maximal rates of expression in relation to one another followingactivation of a cell. In addition, using different RDEs one can programthe transgenes to express a desired amount of transgene encodedpolypeptide and/or a desired amount of AUC or exposure to thepolypeptide encoded by the transgene. Thus, RDEs can be used to providecontrol that produces desired amounts of different transgenepolypeptides at a different (or the same) desired times.

This temporal control can be used to provide desired timing for theproduction of different transgene polypeptides within a cell. Using thistemporal control, a cell can be programmed to express a first transgenethat alters the state of the cell so that is prepared to be affected bythe polypeptide of a second transgene that is expressed at a later time.For example, the first expressed polypeptide could induce the cell tomake and store cytotoxic polypeptides (e.g., granzymes and/or perforins)and the second expressed polypeptide could be involved in the release ofthe cytotoxic polypeptides. Another example of temporal expressioninvolves it use to program a cell to undergo changes (e.g.,differentiation or changing a state of the cell) that requires temporalexpression of two or more gene products. RDEs can be used to mimic thistemporal expression allowing one to control when the cell changes itsstate or differentiates (e.g., programmed differentiation of stemcells). In a stem cell example, the temporal and induction control canbe used to program a stem cell to differentiate when (and where) it isdesired to have the stem cell differentiate into a desired cell type.

The temporal control can also be used to provide desired timing of theproduction of different transgene polypeptides outside of the cell.Using this temporal control, a cell can be activated and secrete a firsttransgene polypeptide that conditions and/or alters a target cell sothat the target cell is prepared to be acted upon by a polypeptideexpressed at later time from a second transgene. For example, the firstpolypeptide could induce a target cell to express a receptor on thetarget cell surface (e.g., FasR, Her2, CD20, CTLA-4, PD-L1, etc.) or apolypeptide in the cell. The first transgene could also induce the cellto secrete a factor that induces the target cell to change its state(e.g., the first transgene could induce the cell to secrete CpG whichcauses the target cell to express OX40 on the target cell surface). Thesecond transgene that reaches maximal rate of expression at a later timecan encode a polypeptide that acts on the induced surface receptor(e.g., FasL, Herceptin, Rituximab, Ipilimumab, Nivolumab, anti-OX40antibody, etc.). The temporal and induction control can also be used tochange the state or differentiation of a target cell by providing to thetarget cell polypeptides in a timed manner where the first polypeptideinduces the target cell to alter its state (e.g., differentiation) sothat it can be acted upon by the second polypeptide (etc. for additionaltransgene polypeptides which reach maximal rate of expression at latertimes).

Autoimmune diseases and other disease states involving an overactiveimmune system (e.g., SARS-CoV-2 infection) can be treated with a AITAMCAR T-cell targeted against autoimmune disease antigen(s). The AITAM CART-cell can include a payload of IL-4, IL-10 or other immunosuppressive.The AITAM CAR T-cell with or without a payload can induce the formationof Tregs that can inhibit the autoimmune disease and/or reduce thetoxicity caused by over-stimulation or chronic stimulation of the immunesystem.

Some examples of diseases and payloads that can be treated using RDEs(Gold elements) with different kinetic parameters (e.g., an RDE thatgives rapid expression early after activation of the cell followed by arapid decline in expression or an RDE that delays expression after cellactivation for 2-3 days) include the following: DLL3 positive cancers(such as IDH1mut gliomas, melanoma, and SCLC) using an anti-DLL3 CAR anda payload of one or more of anti-4-1BB antibody, anti-CD11b antibody,anti-CTLA4 antibody, anti-IL1b antibody, a BiTE, CCL2, anti-CXCR4antibody, anti-CXCL12 antibody, HAC, heparinase, hyaluronidase, Hsp60,Hsp70, IL-2, IL-12, IL-15, IL-18, INFγ, miRNA (e.g., mir155), CD40ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12, CXCR3+CXCL9, CXCL9, ACLY,antagonists of CSF1 receptor, miRNA for Tox (e.g., hsa-mir-26b-5p(MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNA for TCF-7 (e.g.,mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p, miR-491-5p,miR-541-3p), and/or anti-CD28 antibody (including full length andfragments such as single chain antibodies). Optionally, the anti-DLL3CAR with an RDE controlled payload is combined or administered insuccession with another therapy as described above. The combined orsequenced therapy can be an ADC where the antibody binds to a tumorassociate antigen, e.g., DLL3. The combination therapy can be providedto a subject prior to, at the same time, or after the administration ofthe anti-DLL3 CAR with an RDE controlled payload. CD19 positivelymphomas (e.g., NHL) using an anti-CD19 CAR and a payload of IL-12, orone or more of anti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4antibody, anti-IL1b antibody, a BiTE, CCL2, anti-CXCR4 antibody,anti-CXCL12 antibody, HAC, heparinase, hyaluronidase, Hsp60, Hsp70,IL-2, IL-15, IL-18, INFγ, miRNA (e.g., mir155), CD40 ligand, ApoE3,ApoE4, TNFα, CCR2, CCR4/CXCL12, CXCR3+CXCL9, CXCL9, ACLY, antagonists ofCSF1 receptor, miRNA for Tox (e.g., hsa-mir-26b-5p (MIRT030248)hsa-mir-223-3p (MIRT054680)), miRNA for TCF-7 (e.g., mIR-192, mIR-34a,miR-133a, miR-138-5p, miR-342-5p, miR-491-5p, miR-541-3p), and/oranti-CD28 antibody (including full length and fragments such as singlechain antibodies). Optionally, the anti-CD19 CAR with an RDE controlledpayload is combined or administered in succession with another therapyas described above. The combined or sequenced therapy can be an ADCwhere the antibody binds to a tumor associate antigen, e.g., CD19. Thecombination therapy can be provided to a subject prior to, at the sametime, or after the administration of the anti-CD19 CAR with an RDEcontrolled payload. AML with onco-CD43 (sialylation mutant) using ananti-onco-CD43 CAR that recognizes the mutated sialylation and a payloadof one or more of anti-CXCL12 antibody, anti-anti-CXCR4 antibody, orIL-12, and/or one or more of anti-4-1BB antibody, anti-CD11b antibody,anti-CTLA4 antibody, anti-IL1b antibody, a BiTE, CCL2, HAC, heparinase,hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA (e.g.,mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies). Optionally, theanti-onco-CD43 CAR with an RDE controlled payload is combined oradministered in succession with another therapy as described above. Thecombined or sequenced therapy can be an ADC where the antibody binds toa tumor associated antigen, e.g., onco-CD43. The combination therapy canbe provided to a subject prior to, at the same time, or after theadministration of the anti-onco-CD43 CAR with an RDE controlled payload.PSCA positive prostate cancer, bladder cancer or pancreatic cancer usingan anti-PSCA CAR and a payload of heparinase or IL-12, and/or one ormore of anti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4 antibody,anti-IL1b antibody, a BiTE, CCL2, anti-CXCR4 antibody, anti-CXCL12antibody, HAC, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ,miRNA (e.g., mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2,CCR4/CXCL12, CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor,miRNA for Tox (e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p(MIRT054680)), miRNA for TCF-7 (e.g., mIR-192, mIR-34a, miR-133a,miR-138-5p, miR-342-5p, miR-491-5p, miR-541-3p), and/or anti-CD28antibody (including full length and fragments such as single chainantibodies). Optionally, the anti-PSCA CAR with an RDE controlledpayload is combined or administered in succession with another therapyas described above. The combined or sequenced therapy can be an ADCwhere the antibody binds to a tumor associated antigen, e.g., PSCA. Thecombination therapy can be provided to a subject prior to, at the sametime, or after the administration of the anti-PSCA CAR with an RDEcontrolled payload. Triple negative breast cancer with a CAR thatrecognizes cancer testis antigen, misfolded or mutant EGFR (associatedwith triple negative breast cancer), and/or folate receptor alphapeptide and a payload of IL-12 and/or one or more of anti-4-1BBantibody, anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1b antibody,a BiTE, CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC,heparinase, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA(e.g., mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies). Optionally, theanti-cancer testis antigen CAR, anti-misfolded or mutant EGFR CAR, oranti-folate receptor alpha CAR with an RDE controlled payload iscombined or administered in succession with another therapy as describedabove. The combined or sequenced therapy can be an ADC where theantibody binds to a tumor associated antigen, e.g., cancer testisantigen, misfolded or mutant EGFR (associated with triple negativebreast cancer), and/or folate receptor alpha peptide. The combinationtherapy can be provided to a subject prior to, at the same time, orafter the administration of the anti-cancer testis antigen CAR,anti-misfolded or mutant EGFR CAR, or anti-folate receptor alpha CARwith an RDE controlled payload. SEZ6 positive small cell lung cancer(SCLC), neuroendocrine cancers (e.g., medullary thyroid cancer), largecell lung cancer (LCLC), and malignant pheochromocytoma with a CAR thatrecognizes SEZ6 and a payload of IL-12 and/or one or more of anti-4-1BBantibody, anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1b antibody,a BiTE, CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC,heparinase, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA(e.g., mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies). Optionally, theanti-SEZ6 CAR with an RDE controlled payload is combined or administeredin succession with another therapy as described above. The combined orsequenced therapy can be an ADC where the antibody binds to a tumorassociated antigen, e.g., SEZ6. The combination therapy can be providedto a subject prior to, at the same time, or after the administration ofthe anti-SEZ6 CAR with an RDE controlled payload. RNF43 positivecolorectal cancer, colon cancer, and endometrial cancers with a CAR thatrecognizes RNF43 and a payload of IL-12 and/or one or more of anti-4-1BBantibody, anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1b antibody,a BiTE, CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC,heparinase, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA(e.g., mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies). Optionally, theanti-RNF43 CAR with an RDE controlled payload is combined oradministered in succession with another therapy as described above. Thecombined or sequenced therapy can be an ADC where the antibody binds toa tumor associated antigen, e.g., RNF43. The combination therapy can beprovided to a subject prior to, at the same time, or after theadministration of the anti-RNF43 CAR with an RDE controlled payload.TnMUC1 positive breast cancer or pancreatic cancer with a CAR thatrecognizes TnMUC1 and a payload of IL-12 and/or one or more ofanti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1bantibody, a BiTE, CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC,heparinase, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA(e.g., mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies). Optionally, theanti-TnMUC1 CAR with an RDE controlled payload is combined oradministered in succession with another therapy as described above. Thecombined or sequenced therapy can be an ADC where the antibody binds toa tumor associated antigen, e.g., TnMUC1. The combination therapy can beprovided to a subject prior to, at the same time, or after theadministration of the anti-TnMUC1 CAR with an RDE controlled payload.Nectin4 positive urothelial cancer, NSCLC, breast cancer, ovariancancer, bladder cancer, pancreatic cancer, and other solid tumors with aCAR that recognizes Nectin4 and a payload of IL-12 and/or one or more ofanti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1bantibody, a BiTE, CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC,heparinase, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA(e.g., mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies). Optionally, theanti-Nectin4 CAR with an RDE controlled payload is combined oradministered in succession with another therapy as described above. Thecombined or sequenced therapy can be an ADC where the antibody binds toa tumor associated antigen, e.g., Nectin4. The combination therapy canbe provided to a subject prior to, at the same time, or after theadministration of the anti-Nectin4 CAR with an RDE controlled payload.EFNA4 positive triple negative breast cancer, ovarian cancer, colorectalcancer, liver cancer, lung cancer, and other solid tumors with a CARthat recognizes EFNA4 and a payload of IL-12 and/or one or more ofanti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1bantibody, a BiTE, CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC,heparinase, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA(e.g., mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies). Optionally, theanti-EFNA4 CAR with an RDE controlled payload is combined oradministered in succession with another therapy as described above. Thecombined or sequenced therapy can be an ADC where the antibody binds toa tumor associated antigen, e.g., EFNA4. The combination therapy can beprovided to a subject prior to, at the same time, or after theadministration of the anti-EFNA4 CAR with an RDE controlled payload.GPC3 positive hepatocellular carcinoma, lung cancer and other solidtumors with a CAR that recognizes GPC3 and a payload of IL-12 and/or oneor more of anti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4antibody, anti-IL1b antibody, a BiTE, CCL2, anti-CXCR4 antibody,anti-CXCL12 antibody, HAC, heparinase, hyaluronidase, Hsp60, Hsp70,IL-2, IL-15, IL-18, INFγ, miRNA (e.g., mir155), CD40 ligand, ApoE3,ApoE4, TNFα, CCR2, CCR4/CXCL12, CXCR3+CXCL9, CXCL9, ACLY, antagonists ofCSF1 receptor, miRNA for Tox (e.g., hsa-mir-26b-5p (MIRT030248)hsa-mir-223-3p (MIRT054680)), miRNA for TCF-7 (e.g., mIR-192, mIR-34a,miR-133a, miR-138-5p, miR-342-5p, miR-491-5p, miR-541-3p), and/oranti-CD28 antibody (including full length and fragments such as singlechain antibodies). Optionally, the anti-GPC3 CAR with an RDE controlledpayload is combined or administered in succession with another therapyas described above. The combined or sequenced therapy can be an ADCwhere the antibody binds to a tumor associated antigen, e.g., GPC3. Thecombination therapy can be provided to a subject prior to, at the sametime, or after the administration of the anti-GPC3 CAR with an RDEcontrolled payload.

In general, any of the above CAR cells with or without an RDE controlledtransgene(s) can be used in combination or administered in successionwith another molecule (e.g., another therapy). For example, the othermolecule can be a polypeptide, lipid, carbohydrate, nucleic acid, smallmolecule drug, antibody, antibody-drug-conjugate, biological drug, orany combination of the foregoing. The antibody drug conjugate (ADC)includes those described herein. The ADC can bind to the same antigen asthe CAR or it can bind to a different antigen. When the ADC and CAR bindto the same antigen, they may bind to the same or different epitopes onthe same antigen. The ADC and CAR therapy (with or without a RDEcontrolled payload) can be provided at the same time, or one can beadministered to a subject before the other. For example, the ADC and CARcan target a tumor associate antigen and the ADC can be administered thesubject first to reduce the tumor burden, and then the CAR therapy isadministered to clear the remaining cancer cells.

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter. Unless otherwise indicated, thedisclosure is not limited to specific procedures, materials, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

EXAMPLES Example 1. Control of T-Cell Effector Activity with an RDE-CAR

A RDE Car is made using the third generation anti-CD19 CAR cassettedescribed in WO 2012/079000, which is hereby incorporated-by-referencein its entirety for all purposes), and the 3′-UTR of the gene encodingIL-2 (NCBI Reference Sequence Number: NM 000586.3), which is herebyincorporated by reference in its entirety for all purposes). A nucleicacid encoding the IL-2 3′-UTR is engineered into the anti-CD19 CARcassette in an appropriate expression vector. The IL-2, 3′-UTR sequenceused was:

(SEQ ID NO: 21) taattaagtgatcccacttaaaacatatcaggccttctATTTATTTAaatATTTAaattttatATTTAttgttgaatgtatggtttgctacctattgtaactattattcttaatcttaaaactataaatatggatcttttatgattctttttgtaagccctaggggctctaaaatggtttcacttATTTAtcccaaaatATTTAttattatgttgaatgttaaatatagtatctatgtagattggttagtaaaactATTTAataaatttgataaatataaa

The anti-CD19 RDE CAR and anti-CD19 CAR constructs are transfected byroutine methods into different populations of T-cells (primary humanT-cells), and stable populations of T-cells are selected usingappropriate antibiotics (or other selection schemes). T-cell populationswith anti-CD19 RDE CARs (CD19⁻/CD22⁻/CD3⁺) and T-cell populations withanti-CD19 CARs (CD19⁻/CD22⁻/CD3⁺) are activated by co-incubation withanti-CD3/CD28 beads and allowed to return to quiescent state afterdebeading.

Quiescent anti-CD19 RDE CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at RDE CAR T-cell:Raji target ratiosof 2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The RDE-CAR T-cells and theRaji cells are grown together for 24 hours. Cultures are washed, andthen stained with anti-CD22 and anti-CD3 reagents, followed by countingof CD22⁺ (Raji target cells) and CD3⁺ cells (Smart CAR T-cells). Thesemeasurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the RDE-CAR T-cells atdifferent levels of RDE-CAR expression.

Activated anti-CD19 RDE CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at RDE CAR T-cell:Raji target ratiosof 2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2, mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The RDE-CAR T-cells and theRaji cells are grown together for 24 hours. Samples from culture mediaare taken and tested for IL-2 by ELISA.

As a control activated anti-CD19 CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The CAR T-cells and the Rajicells are grown together for 24 hours. Cultures are washed, and thenstained with anti-CD22 and anti-CD3 reagents, followed by counting ofCD22⁺ (Raji target cells) and CD3⁺ cells (CAR T-cells).

As a control, activated anti-CD19 CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The CAR T-cells and the Rajicells are grown together for 48 hours. Samples from culture media aretaken and tested for IL-2 by ELISA.

Example 2: Removal of MicroRNA Binding Sites from an RDE

The AU-rich element from the 3′-UTR of IL-2 has mir-181 and mir 186microRNA binding sites. Different combinations of the microRNA siteswere removed from the 3′-UTR of IL-2. When the MIR186 micro-RNA siteswere removed from the 3′-UTR of IL-2 the dynamic range of expressionfrom constructs with this UTR increased 50 fold. The modified IL-2,3′-UTR replaces CTT in the sequence with GAA and is shown below (the newGAA is underlined in the sequence):

(SEQ ID NO: 22) taattaagtgatcccacttaaaacatatcaggccttctATTTATTTAaatATTTAaattttatATTTAttgttgaatgtatggtttgctacctattgtaactattattcttaatcttaaaactataaatatggatcttttatgattGAAtttgtaagccctaggggctctaaaatggtttcacttATTTAtcccaaaatATTTAttattatgttgaatgttaaatatagtatctatgtagattggttagtaaaactATTTAataaatttgataaatataaa 

The AU-rich element from the 3′UTR of IFNg also has micro-RNA bindingsites characterized as mir-125. The sequence of the IFNg RDE is:

(SEQ ID NO: 23) tggttgtcctgcctgcaatatttgaattttaaatctaaatctATTTAttaatATTTAacattATTTAtatggggaatatatttttagactcatcaatcaaataagtATTTAtaatagcaacttttgtgtaatgaaaatgaatatctattaatatatgtattATTTAtaattcctatatcctgtgactgtctcacttaatcctttgttttctgactaattaggcaaggctatgtgattacaaggetttatctcaggggccaactaggcagccaacctaagcaagatcccatgggttgtgtgtttatttcacttgatgatacaatgaacacttataagtgaagtgatactatccagttactgccggtttgaaaatatgcctgcaatctgagccagtgctttaatggcatgtcagacagaacttgaatgtgtcaggtgaccctgatgaaaacatagcatctcaggagatttcatgcctggtgcttccaaatattgttgacaactgtgactgtacccaaatggaaagtaactcatttgttaaaattatcaatatctaatatatatgaataaagtgtaagttcacaacta

Different combinations of the micro-RNA sites were removed from the3′UTR of IFNg and tested for increased expression. When the mir125micro-RNA sites were removed from the 3′-UTR of IFN-γ the expressionrate from constructs with this UTR is increased.

Expression of GFP in T-cells, transfected with the RDE-GFP plus themicroRNA sites, is compared to expression of GFP in T-cells with theRDE-GFP in which the microRNA sites have been removed, followingactivation with CD3/CD28 beads for 24 hours. The removal of the microRNAsites increased expression of the GFP by a factor of between 2-5 after24 hours, relative to the cells with microRNA sites.

Example 3: Payload Delivery to DLBCL Using an Anti-CD19 CAR T-Cell

The anti-CD19 Smart CAR T-lymphocytes and anti-CD19 CAR T-celllymphocytes of Example 6 are used in this example. These CART-lymphocytes are further engineered to include a construct encoding aPD-1 inhibitor under the control of the 3′-UTR of IL2 that has beenmodified by removal of the MIR186 sites. PD-1 inhibitors expressed bythe construct include, for example, Pembrolizumab (Keytruda®), Nivolumab(Opdivo®), Cemiplimab (Libtayo®), Atezolizumab (Tecentriq®), Avelumab(Bavencio®), Durvalumab (Imfinzi®), BMS-936558, Lambrolizumab, orpolypeptides derived from these drugs. Other PD-1 inhibitors that may beexpressed by the construct include those disclosed in Herbst et al., JClin Oncol., 31:3000 (2013); Heery et al., J Clin Oncol., 32:5s, 3064(2014); Powles et al., J Clin Oncol, 32:5s, 5011(2014); Segal et al., JClin Oncol., 32:5s, 3002 (2014), or U.S. Pat. Nos. 8,735,553; 8,617,546;8,008,449; 8,741,295; 8,552,154; 8,354,509; 8,779,105; 7,563,869;8,287,856; 8,927,697; 8,088,905; 7,595,048; 8,168,179; 6,808,710;7,943,743; 8,246,955; and 8,217,149.

T-cell populations with anti-CD19 Smart CARs/PD-1 (CD19-/CD22-/CD3+) andT-cell populations with anti-CD19 CARs/PD-1 (CD19-/CD22-/CD3+) areactivated by co-incubation with anti-CD3/CD28 beads. T-cells withanti-CD19 Smart CARs/PD-1 inhibitor or anti-CD19 CARs/PD-1 inhibitorwere incubated with theophylline at 0, 75 and 250 μM for 72 hours.Activated anti-CD19 Smart CAR/PD-1 T-cells or anti-CD19 CAR/PD-1 T-cellswere co-cultured with CD19⁺/CD22⁺/CD3− Raji target cells at SmartCAR/PD-1 T-cell:Raji target ratios of 2:1, 5:1, and 10:1. Ligand for theRNA control device, theophylline is maintained in the culture medium atconcentrations of 0 μM, 75 μM, and 250 μM. The Smart-CAR/PD-1 T-cells orCAR/PD-1 T-cells and the Raji cells are grown together for 18 hours.Cultures are washed, and then stained with anti-CD22 and anti-CD3reagents, followed by counting of CD22+(Raji target cells) and CD3+cells (Smart CAR T-cells). Samples from culture media are also taken at6, 12 and 18 hours, and tested for PD-1 inhibitor by ELISA.

Example 4: Payload Delivery to AML Using an Anti-CD133 CAR T-Cell

A CAR is made using the anti-CD20 CAR cassette described in Budde 2013(Budde et al. PLoS1, 2013 doi:10.1371/journal.pone.0082742, which ishereby incorporated-by-reference in its entirety for all purposes), withthe anti-CD133 mAb 293C3-SDIE is used for the extracellular element(Rothfelder et al., 2015,ash.confex.com/ash/2015/webprogram/Paper81121.html, which isincorporated by reference in its entirety for all purposes) replacingthe anti-CD20 extracellular domain. The anti-CD133 CAR also can encodethe RNA control device, 3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad.Sci. 104 (36): 14283-88, which is hereby incorporated by reference inits entirety for all purposes). A nucleic acid encoding the anti-CD20CAR cassette is engineered to replace the anti-CD20 extracellular domainwith the anti-CD133 element, and optionally the RNA control device isalso engineered into the cassette. The anti-CD133 CAR with or withoutthe RNA control device are cloned into appropriate expression vectors.

These anti-CD133 CAR and anti-CD133 Smart CAR constructs are transfectedby routine methods into T-lymphocytes (Jurkat cells and/or primary humanT-lymphocytes), and stable populations of T-lymphocytes are selectedusing appropriate antibiotics (or other selection schemes).

These CAR T-lymphocytes are further engineered to include a constructencoding a PD-1 inhibitor under the control of the RDE from the 3′-UTRof IL2 that has been modified by removal of a MIR186 site. PD-1inhibitors expressed by the construct include, for example,Pembrolizumab (Keytruda®), Nivolumab (Opdivo®), Cemiplimab (Libtayo®),Atezolizumab (Tecentriq®), Avelumab (Bavencio®), Durvalumab (Imfinzi®),BMS-936558, Lambrolizumab, or polypeptides derived from these drugs.Other PD-1 inhibitors that may be expressed by the construct includethose disclosed in Herbst et al., J Clin Oncol., 31:3000 (2013); Heeryet al., J Clin Oncol., 32:5s, 3064 (2014); Powles et al., J Clin Oncol,32:5s, 5011(2014); Segal et al., J Clin Oncol., 32:5s, 3002 (2014), orU.S. Pat. Nos. 8,735,553; 8,617,546; 8,008,449; 8,741,295; 8,552,154;8,354,509; 8,779,105; 7,563,869; 8,287,856; 8,927,697; 8,088,905;7,595,048; 8,168,179; 6,808,710; 7,943,743; 8,246,955; and 8,217,149.

T-lymphocyte populations with anti-CD133 CAR/PD-1 inhibitor oranti-CD133 Smart CAR/PD-1 inhibitor (CD20⁻/CD22⁻/CD3⁺) are activated byco-incubation with anti-CD3/CD28 beads.

Activated anti-CD133 CAR/PD-1 inhibitor or anti-CD133 Smart CAR/PD-1inhibitor T-lymphocytes are co-cultured with CD133⁺/CD3⁻ AML targetcells (e.g., U937, MV4-11, MOLM-14, HL-60 and/or KG1a) at anti-CD133 CARand/or anti-CD133 Smart CAR T-lymphocyte:AML target ratios of 2:1, 5:1,and 10:1. Ligand for the RNA control device, theophylline, is added tothe culture medium at concentrations in the range of 500 μM to 1 mM(lower or greater concentrations can be used to titrate Smart-CARactivity to the desired level). The anti-CD133 CAR/PD-1 inhibitor and/oranti-CD133 Smart CAR/PD-1 inhibitor T-lymphocytes and the AML cells aregrown together for 48 hours. Cultures are washed, and then stained withanti-CD133 and anti-CD3 reagents, followed by counting of CD133⁺ (AMLtarget cells) and CD3⁺ cells (anti-CD133 CAR, anti-CD133 DE-CAR,anti-CD133 Smart CAR, and/or the anti-CD133 DE-Smart CAR T-lymphocytes).These measurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the anti-CD133 CAR/PD-1inhibitor and/or anti-CD133 Smart CAR/PD-1 inhibitor T-lymphocytes atdifferent levels of CAR expression. Samples from culture media are alsotaken at 12, 24, 26 and 48 hours, and tested for PD-1 inhibitor byELISA.

Example 5: An RDE Construct for Expressing a Second Transgene

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a GFP-RDE1 (3′-UTR from IFNg) insert. These twoinserts/cassettes were placed in the same lenti virus construct. Theanti-CD19 CAR cassette and the insert with the GFP-RDE are transcribedin opposite directions, and the control regions for each are located inbetween the two insert/cassettes. The control region for the GFP-RDEinsert was MinP and the RDE was the endogenous 3′-UTR of IFNg. Thecontrol region of the anti-CD19 CAR cassette was MND (as describedabove). CD4+ T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cells oranti-CD³/anti-CD28 beads were incubated with the transduced T cells for24h. At 24h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and GFP expression in the T cells.

The transduced T-cells showed an increase in fluorescence when culturedwith Raji target cells (activate CAR) of 1.0% to 6.5% (about 6.5 fold),and increase in fluorescence when cultured with CD3/CD28 beads (activateTCR) of 1.0% to 4.4% (about 4.4 fold). The transformed T-cells showed achange in activated cells in the population when cultured with Rajicells of 0.9% to 84.8%, and when cultured with CD3/CD28 beads of 0.9% to90.8%.

Example 6: A Modified RDE2 Construct for Expressing a Second Transgene

Constructs were made using an anti-CD19 CAR cassette as described inExamples 11 and 12, and a GFP-RDE2.1 (IL-2 RDE) insert. The RDE2.1 wasmodified to remove the MIR186 microRNA sites, altering nucleotides fromthe 3′-UTR of IL-2 which was used as RDE2.

These two inserts/cassettes were placed in the same lenti virusconstruct. The anti-CD19 CAR cassette and the insert with the GFP-RDEare transcribed in opposite directions, and the control regions for eachare located in between the two insert/cassettes. The control region forthe GFP-RDE insert was a MinP. The control region of the anti-CD19 CARcassette in was MND (as described above). CD4+ T-cells were transducedwith the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cells oranti-CD³/anti-CD28 beads were incubated with the transduced T cells for24h. At 24h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and GFP expression in the T cells.

The transduced T-cells showed a change in activated cells in thepopulation when cultured with Raji cells of 3.9% to 12.1%, and whencultured with CD3/CD28 beads of 3.9% to 11.1%.

Example 7: An RDE Construct for Expressing a Luciferase Transgene

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a Luciferase-RDE1 (3′-UTR of IFNg, Gold1) insertor a Luciferase-3′-UTR (a 3′-UTR that does not confer differentialtransgene translation in response to metabolic state of the cell,3′-UTR). The anti-CD19 CAR cassette and the insert with theluciferase-RDE1 are transcribed in opposite directions, and the controlregions for each are located in between the two insert/cassettes. Thecontrol region for the Luciferase-RDE1 insert and Luciferase-3′-UTR wereeither a MinP promoter or an NFAT promoter having the sequences of:

(MinP) SEQ ID NO: 24 TAGAGGGTATATAATGGAAGCTCGACTTCCAG (NEAT)SEQ ID NO: 25 GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGACTCTAGAGGGTATATAATGGAAGCTCGAATTCThe control region of the anti-CD19 CAR cassette was the MND promoter.CD4+ T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cells oranti-CD³/anti-CD28 beads were incubated with the transduced T cells for24h. At 24h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and luciferase expression in the T cells.

FIG. 2 shows that the transduced T-cells had an increase inbioluminescence when cultured with Raji target cells (activate CAR) orwhen cultured with CD3/CD28 beads (activate TCR) as compared tobioluminescence of T-cells at resting. The T-cells with a NFAT promoterand the 3′-UTR of IFNg (Gold1) showed a larger on-off response from CARstimulation versus TCR stimulation. Under all conditions, T-cells withGold1 had lower amounts of bioluminescence than T-cells under the sameconditions (and same promoter) with Luciferase that was not controlledby the 3′UTR of IFNg (3′-UTR).

Example 8: Comparison of RDEs Controlling Luciferase

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a Luciferase-RDE1 (3′ UTR of IFNg, Gold1) insert,a Luciferase-RDE2 (3′-UTR of IL-2, Gold2) insert, a Luciferase-RDE3(3′-UTR of IL-2 modified as described above to remove the mir186 sites,Gold3), or a Luciferase-3′-UTR (a 3′-UTR that does not conferdifferential transgene translation in response to metabolic state of thecell, 3′-UTR). Combinations of these inserts/cassettes shown in FIG. 3were placed in the similar lenti virus constructs. The anti-CD19 CARcassette and the insert with the luciferase-RDE are transcribed inopposite directions, and the control regions for each are located inbetween the two insert/cassettes. The control region for theLuciferase-RDE insert and Luciferase-3′-UTR were either a MinP promoteror an NFAT promoter. The control region of the anti-CD19 CAR cassettewas the MND promoter, and CD4+ T-cells were transduced with thebicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set CD19+ Raji B cells were incubated with the transduced Tcells for 24h. At 24h, the T cells were stained for CD25 and CD69, whichare activation markers, and subject to flow cytometry to measure thesemarkers and luciferase expression in the T cells.

FIG. 3 shows that the transduced T-cells had an increase inbioluminescence when cultured with Raji target cells (activate CAR) ascompared to bioluminescence of T-cells at resting for constructs withRDE1 (Gold1), RDE2 (Gold2), or RDE3 (Gold3). The T-cells with a NFATpromoter and the RDE1 showed a larger on-off response than T-cells witha MinP promoter and the corresponding RDE. Under all conditions, T-cellswith an RDE controlling luciferase had lower amounts of bioluminescencethan T-cells with luciferase cassettes that were not controlled by anRDE. Combined with the MinP promoter, RDE1 gave a 4.1-fold increase inbioluminescence with CAR stimulation, RDE2 gave a 1.8-fold increase inbioluminescence, and RDE3 gave a 1.4-fold increase. Combined with theNFAT promoter, RDE1 gave a 8.5-fold increase in bioluminescence with CARstimulation, RDE2 gave a 3.1-fold increase in bioluminescence, and RDE3gave a 1.3-fold increase. With either promoter, the RDE3 construct gavethe highest amount of bioluminescence, the RDE1 construct gave thelowest amount of bioluminescence, and the RDE2 construct gave an amountof bioluminescence between RDE3 and RDE1.

Example 9: An RDE Construct for Expressing IL-12

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and an IL-12-RDE1 (3′-UTR of IFNg) insert or an IL-123′-UTR (a 3′-UTR that does not confer differential transgene translationin response to metabolic state of the cell). The anti-CD19 CAR cassetteand the insert with the IL-12-RDE1 are transcribed in oppositedirections, and the control regions for each are located in between thetwo insert/cassettes. The control region for the IL-12-RDE1 insert andIL-12 3′-UTR were either a minP promoter or an NFAT promoter. Thecontrol region of the anti-CD19 CAR cassette was the MND promoter. CD4+T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set, CD19+ Raji B cells were incubated with the transduced Tcells for 24h. At 24h, the T cells were stained for CD25 and CD69, whichare activation markers, and subject to flow cytometry to measure thesemarkers. IL-12 expression in the T cells was measured by ELISA.

FIG. 4 shows that the transduced T-cells had an increase in IL-12expression when cultured with Raji target cells (activate CAR) ascompared to IL-12 expression of T-cells at resting using constructscontrolled by the MinP promoter or NFAT promoter. T-cells with the NFATpromoter and RDE1 (Gold1) showed a 168-fold change in IL-12 expressionform resting to CAR stimulation. T-cells with the NFAT promoter and a3′-UTR (not responsive to CAR stimulation, 3′-UTR) showed a 50-foldchange in expression, and a minP promoter with RDE1 (Gold1) showed a 6.3fold change in expression.

Example 10: AU Elements and Steady State Expression

Constructs were made with different RDEs operably linked to a nucleicacid encoding luciferase. The different RDEs used were AU 4 (CTLA4), AU13 (IL-5), AU 14 (IL-6), AU 15 (IL-9), AU 16 (IL-10), AU 17 (IL-13), andAU 101 (IFNg). These luciferase-AU constructs were transduced intoprimary T-cells. After the cells returned to the resting stage they wereplated and sham induced (basal) or induced with anti-CD3 and anti-CD28antibody (activated). At 24 hours post activation the amount ofluciferase units in each was measured. These amounts are plotted in thebar graph of FIG. 5.

The AU elements in this example had different basal expression levels,different induced expression levels (at 24 hours), and different levelsof fold induction. The AU constructs showed different amounts of basalexpression, different amounts of induced expression and differentamounts of fold induction (or dynamic range).

Example 11: AU Elements and Expression Parameters

Constructs were made with different RDEs operably linked to a nucleicacid encoding luciferase. The different RDEs used were AU 2 (CSF2), AU 3(CD247), AU 5 (EDN1), AU 7 (SLC2A1), AU 10 (Myc), AU 19 (TMEM-219), AU20 (TMEM-219snp), AU 21 (CCR7), AU 22 (SEM-A4D), AU 23 (CDC42-SE2), andAU 101 (IFNg). These luciferase-AU constructs were transduced intoprimary T-cells. After the cells returned to the resting stage they wereplated and either not treated (basal) or activated with anti-CD3 andanti-CD28 antibody (activated). At 24 hours post activation the amountof luciferase units in each was measured. These amounts are plotted inthe bar graph of FIG. 6. Alternatively, after the cells returned toresting stage they were plated into 96-well plates in quadruplicate formeasuring at each time point: 1 day, 3 days, 6 days and 8 days. Thecells were either not treated or activated with anti-CD3 and anti-CD28antibody, and luciferase activity was measured at 1 day, 3 days, 6 daysand 8 days. These results are plotted in the bar graph of FIG. 7, andshown in Table 1 below. FIG. 8 shows selected data plotted in a bargraph. The numbers in parentheses in Table 1 below are the LuciferaseUnits on Days 3, 6, and 8 divided by the Luciferase Units of Day 1.

TABLE 1 Luciferase Units AU Construct Day 1 Day 3 Day 6 Day 8 AU 2(CSF2) 60051 306035 (5) 578305 (10) 591953 (10) AU 101 (IFNg) 85816473395 (6) 724129 (8) 817447 (10) AU 5 (EDN1) 69391 613921 (9) 838040(12) 1023000 (15) AU 3 (CD247) 44939 595753 (13) 961839 (21) 1116000(25) AU 20 (TMEM-219snp) 1135000 10750000 (9) 21020000 (19) 25480000(22) AU 10 (Myc) 1233000 16020000 (13) 26780000 (22) 27800000 (23) AU 7(SLC2A1) 4914 80906 (16) 132974 (27) 136537 (28) AU 21 (CCR7) 27128465140 (17) 604016 (22) 692715 (26) AU 23 (CDC42-SE2) 71105 1215000 (17)2012000 (28) 2110000 (30) AU 22 (SEM-A4D) 226815 2829000 (12) 6106000(27) 7396000 (33) AU 19 (TMEM-219) 833146 11260000 (14) 22560000 (27)27500000 (33)

The AU elements in FIG. 6, FIG. 7 and Table 1 had different basalexpression levels, different induced expression levels (at 24 hours),and different levels of fold induction. The basal expression levelsdiffered over an about 2000 fold range for these AU elements (AU 7 to AU20), and the induced expression levels differed over an about 5500 foldrange (AU 7 to AU 10). Basal expression for the constructs ranged from1390 for AU 7 (SLC2A1) to 2,927,000 for AU 20 (TMEM-219snp). Activatedexpression ranged from 4914 for AU 7 (day 1) to 27,800,0000 for AU 10(day 8). FIG. 8 and Table 1 show that some AU elements had lower levelsof output, for example, AU 101 (IFNg), AU 2 (CSF2), AU 5 (EDN1), AU 7(SLC2A1), AU 21 (CCR7), and AU 23 (CDC42-SE2). Some AU elements hadintermediate amounts of output: AU 19 (TMEM-219) and AU 22 (SEM-A4D).And some AU element had high output: AU 20 (TMEM-219snp) and AU 10(Myc).

The Luciferase data was also analyzed for dynamic range (fold inductionor luciferase activated/luciferase basal) of each luciferase-AUconstruct. The dynamic range (fold induction) for each AU construct atDays 1, 3/4 (activated expression was measured on Day 3 and basalexpression was measured on Day 4), 6 and 8. This data is shown below inTable 2, and plotted in bar graphs in FIG. 9 and FIG. 10.

TABLE 2 Fold Induction AU Construct Day 1 Day 3/4* Day 6 Day 8 AU 2 9.39.3 8.1 5.8 AU 101 16.9 14.6 12.3 10.5 AU 5 5.7 11.9 10.1 9.4 AU 21 5.627.3 28.6 29.6 AU 3 1.8 6.7 8.1 6.8 AU 20 3.1 7.3 9.3 8.7 AU 10 3.7 13.214.1 10.2 AU 7 3.5 12.3 13.1 10.6 AU 23 2.6 12.8 15.1 12.2 AU 19 2.3 9.312.9 13.8 AU 22 1.2 4.6 6.9 7.1 *Induction was measured on Day 3 andbasal was measured on Day 4.

At Day 1 dynamic range (fold induction=activated/basal) ranged fromabout 1 (AU 22) to about 17 (AU 101). At Day 3/4, dynamic range variedfrom about 4.5 (AU 22) to about 27 (AU21). At Day 6, dynamic rangevaries from about 7 (AU 22) to about 29 (AU 21). On Day 8, dynamic rangevaried from about 7 (AU22) to about 30 (AU 21). The AU constructs showeda number of related patterns. AU 2 and AU 101 showed a rapid increase indynamic range on Day 1, and then the dynamic range decreased on days 6and 8. AU 5 and AU 21 show increasing dynamic range from day 0 to day3/4, and then the dynamic range is maintained through days 6 and 8. AU3, AU 20, AU 10, AU 7 and AU 23 showed rising dynamic range from day 0to day 6, and then the dynamic range decreased on day 8. AU 19, and AU22, showed rising dynamic ranges from day 0 to day 8.

AU 21 and AU 23 showed accelerating dynamic range and these AUconstructs also had low basal expression (day 1=4865 and 27363,respectively). AU 2 and AU 101 showed decreasing dynamic range from 24hours to 72 hours and these AU elements also had low basal expression.AU 5 and AU 20 also showed decreasing dynamic range from day 1 to day3/4 (though more expression than AU 2 and AU 101) and AU 5 had low basalexpression whereas AU 20 had high basal expression. AU 10, AU 19 and AU22 showed consistent dynamic range from day 1 to day 3/4 and had highbasal levels of expression. AU 3 and AU 7 also had consistent dynamicrange from day 1 to day 3/4 and had low basal expression levels.

The above data shows that different AU elements have different temporaleffects on expression from days 1-8. Some AU elements show acceleratingdynamic range over different portions of the time range. The AU elementsshow different amounts of total expression (C_(max)) and different timesto maximum expression (T_(max)). The AU elements also show differentmaximum dynamic ranges and time to reach these maximums. These differingkinetics of expression can be used to provide customized basal, C_(max),T_(max), dynamic range, and time to max dynamic range for a desiredtransgene. These differing kinetics can also be used to providetemporally distinct expression for two transgenes in a cell afteractivation of the cell.

Example 12: AU Element Control with Glucose and Galactose

Constructs were made with different RDEs operably linked to a nucleicacid encoding luciferase. The RDE was an AU element responsive toglycolytic state of the cell. The AU element—luciferase constructs weretransduced into T-cells. After the cells reached the resting state, theywere split into wells and fed media including either glucose orgalactose. Luciferase activity was measured on days 3 and 5. Theseresults are shown in the bar graph of FIG. 11. The results show thatglucose increased expression of luciferase compared to galactose and theamount of expression increased from days 3 to 5. On day 3 the glucosetreated cells had 15× more expression of luciferase than the galactosetreated cells and on day 5 this had grown to 27× more expression.

Example 13: Delivery and Design of a Viral Payload

A Smart Car is made using the third generation anti-CD19 CAR cassettedescribed in WO 2012/079000, which is hereby incorporated-by-referencein its entirety for all purposes), and the RNA control device,3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad. Sci. 104 (36):14283-88, which is hereby incorporated by reference in its entirety forall purposes). A nucleic acid encoding the 3XL2bulge9 control device isengineered into the anti-CD19 CAR cassette in an appropriate expressionvector. The anti-CD19 Smart CAR and anti-CD19 CAR constructs aretransfected by routine methods into different populations of T-cells(Jurkat cells and/or primary human T-cells), and stable populations ofT-cells are selected using appropriate antibiotics (or other selectionschemes). T-cell populations with anti-CD19 Smart CARs(CD19⁻/CD22⁻/CD3⁺) and T-cell populations with anti-CD19 CARs(CD19⁻/CD22⁻/CD3⁺) are activated by co-incubation with anti-CD3/CD28beads.

Third generation Lentiviral packaging, envelope, and transfer plasmidsare obtained from addgene. The Rev encoding packaging plasmid isengineered to include the AU101 (INFg) RDE in the 3′-UTR of Rev. Themodified Rev packaging plasmid, the Gag Pol packaging plasmid, and theenvelope plasmid are transfected into anti-CD19 T-lymphocyte cells. Atransfer plasmid is engineered to include GFP as the transgene in thetransfer plasmid. This transfer plasmid is also transfected into theanti-CD19 CAR T-lymphocyte cells.

Anti-CD19 Smart CAR T-lymphocytes are co-cultured withCD19⁺/CD22⁺/CD3-Ramos target cells at Smart CAR T-lymphocyte:Raji targetratios of 2:1, 5:1, and 10:1. Ligand for the RNA control device,theophylline is added to the culture medium at concentrations in therange of 2 μM to 2 mM (2 μM, 10 μM, 20 μM, 100 μM, 200 μM, 1 mM, and 2mM). The Smart-CAR T-cells and the Raji cells are grown together for 48hours.

At the end of the incubation period, the culture media is separated fromthe T-lymphocytes and Raji cells. Viral titer in the supernatant ismeasured using an ELISA with anti-lentivirus antibody reagents.Infectivity and payload delivery by the viruses is tested by infectingHEK 293 cells with the virus, and after a suitable incubation timemeasuring GFP fluorescence from the transduced HEK 293 cells.

Example 14: Payload Delivery Using Gold in a Mouse Lymphoma Model

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes, and a Luciferase-AU (3′ UTR of IL-6) insert. Theseconstructs were placed in a bicistronic lenti virus construct. Theanti-CD19 CAR cassette and the insert with the luciferase-RDE aretranscribed in opposite directions on the bicistronic vector, and thecontrol regions for each are located in between the twoinsert/cassettes. The control region for the Luciferase-RDE insert was aMinP promoter. The control region of the anti-CD19 CAR cassette was theMND promoter. CD4+ T-cells were transduced with the bicistronicconstruct.

A second construct was made using the anti-CD19 CAR cassette describedabove and a Luciferase insert (without the RDE element so thatexpression was constitutive). Both constructs were separately transducedinto different groups of T-cells.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24h alone in medium. For the ‘Rajico-culture’ set CD19+ Raji B cells were incubated with the transduced Tcells for 24h. At 24h, the T cells were stained for CD25 and CD69, whichare activation markers, and subject to flow cytometry to measure thesemarkers and luciferase expression in the T cells. These in vitro resultsshowed that the anti-CD19 CAR T-cells made luciferase after activationof the T-cells through the CAR.

These anti-CD19 CAR T-cells with the luciferase-RDE were also tested ina mouse model for lymphoma. CD19+ Raji cells were implanted in theflanks of NSG mice. After tumor formation, the anti-CD19 CAR T-cellswere injected into the mice and the mice were scanned for luminescence.Imaging of the mice showed luminescence at the tumor sites fromanti-CD19 CAR T-cells that have been activated by the CD19 positivetumor. The amount of luminescence increased over time as more T-cellswere activated. In contrast, the anti-CD19 CAR T-cells with constitutiveexpression of luciferase should luminescence throughout the mice as wellas at the site of the tumors in the flanks of the mice.

Example 15: Payload Delivery to αvβ6 Positive Solid Tumor

A nucleic acid encoding a knottin as described in Silverman et al., J.Mol. Biol. 385:1064-75 (2009) and Kimura et al, Proteins 77:359-69(2009), which are incorporated by reference in their entirety for allpurposes is operably linked to a nucleic acid encoding the CARcomponents aCD43z,CD8Hinge,CD8transmembrane,41BB(CD28 or othercostim),and CD3z to make a nucleic acid encoding an anti-αvβ6 CAR.

The nucleic acid encoding the anti-αvβ6 CAR is transfected by routinemethods into T-cells (Jurkat cells and/or primary human T-cells), andstable populations of T-cells are selected using appropriate antibiotics(or other selection schemes). T-cell populations with anti-αvβ6 CARs areactivated by co-incubation with anti-CD3/CD28 beads. These cells arealso engineered with an expression cassette encoding IL-12 operablylinked to the Gold element from INFg or AU 21 (CCR7) is placed under thecontrol of the promoter Min P.

The anti-αvβ6 CAR T-cells are incubated in wells with αvβ6 tumor cells.After incubation, the wells are tested for secretion of IL-12 from theanti-αvβ6 CAR T-cells. anti-αvβ6 CAR T-cells secrete IL-12 whenincubated with αvβ6 tumor cells, and the controls show low or nosecretion when the CAR T-cell is not stimulated.

Example 16: An Anti-Onco CD 43 CAR for AML

A single chain antibody for onco-sialylated CD 43 was made using ananti-onco-sialylated CD 43 antibody. The nucleic acid encoding thissingle-chain antibody was combined with a nucleic acid encoding the CARcomponents aCD43z,CD8Hinge,CD8transmembrane,41BB(CD28 or othercostim),and CD3z to make a nucleic acid encoding an anti-onco-sialylatedCD 43 CAR.

The nucleic acid encoding the anti-onco-sialylated CD 43 CAR istransfected by routine methods into T-cells (Jurkat cells and/or primaryhuman T-cells), and stable populations of T-cells are selected usingappropriate antibiotics (or other selection schemes). T-cell populationswith anti-onco-sialylated CD 43 CARs are activated by co-incubation withanti-CD3/CD28 beads.

Example 17: Payload Delivery to CD 43 Positive AML

An expression cassette encoding IL-12 operably linked to the Goldelement from INFg or AU 21 (CCR7) is placed under the control of thepromoter Min P, and engineered into the anti-onco-sialylated CD 43 CART-cell.

The anti-onco-sialylated CD 43 CAR T-cells are incubated in wells withAML cells. After incubation, the wells are tested for secretion of IL-12from the anti-onco-sialylated CD 43 CAR T-cells. Anti-onco-sialylated CD43 CAR T-cells secrete IL-12 when incubated with AML cells, and thecontrols show low or no secretion when the CAR T-cell is not stimulated.

Example 18: miRNA as a Payload

A payload transgene encoding IL-12 is engineered to have an artificialintron encoding a mir155 cassette as disclosed in Du et al., FEBsJournal 273:5421-5427 (2006) or Chung et al., Nucl Acids Res 34:e53(2006). The mir155 cassette is engineered to include an AU element suchas, for example, AU101 (IFNg) or AU14 (IL-6), operably linked to it, andthe transgene is also engineered with an AU element such as AU101 orAU14. This transgene with the mir155 intron is engineered into primaryT-cells. An anti-CD19 CAR as described in Example 14 is also engineeredinto the primary T-cells.

The anti-CD19 CAR T cells with the IL-12 payload are allowed to returnto resting state, and then are tested after stimulation as follows. Forthe ‘no stimulation’ set, transduced T-cells are incubated for 24h alonein medium. For the ‘Raji co-culture’ set CD19+ Raji B cells areincubated with the transduced T cells for 24h. At 24h, the T cells arestained for CD25 and CD69, which are activation markers, and subject toflow cytometry to measure these markers. The cells are also tested forexpression of the payload IL-12.

These anti-CD19 CAR T-cells with the IL-12 payload are also tested in amouse model for lymphoma. CD19+ Raji cells are implanted in the flanksof NSG mice. After tumor formation, the anti-CD19 CAR T-cells with theIL-12 payload are injected into the mice. At every third day starting atday 4 after administration, tumor killing in the mice is measured usingcalipers.

Example 19: IL-12 Payload Delivery

A construct with an anti-CD19 CAR as described in Example 14 was made. Aconstruct with the NFAT promoter operably linked to a nucleic acidencoding IL-12 followed by AU101 (the RDE from INFg) was also made. TheIL-12 transcript made from the construct operably links the codingsequence for IL-12 to the AU101 RDE. A second IL-12 construct was madethat provided constitutive expression of IL-12. A third construct placedLuciferase under control of an AU14 (IL-6).

The constructs were transduced into primary T-cells which were thenallowed to return to a resting state. This produced anti-CD19 CART-cells with payloads of IL-12 (RDE controlled or constitutive) orluciferase.

The primary T-cells with the anti-CD19 CAR and IL-12 payload (RDEcontrolled or constitutive) or luciferase payload were administered tomice bearing CD19+ tumors in their flanks. Killing of tumor cells wasmonitored over 42 days. The mice which received T-cells with theanti-CD19 CAR and luciferase payload showed a moderate amount of tumorcell killing (about 3 logs). The mice receiving the IL-12 payloads had alarge amount of tumor cell killing (6-7 logs). A comparison of IL-12serum levels in the mice receiving the constitutive or AU101 controlledIL-12 had 10-fold differences in the systemic IL-12 levels with theAU101 controlled payload having 10 times lower amounts of IL-12 than theconstitutive IL-12 payload.

The RDE control of IL-12 expression lowered systemic IL-12 levels in themice but gave localized concentrations of IL-12 that improved tumor cellkilling. After the activated CAR T-cells kill the tumor cells these CART-cells can migrate from the tumor site to lymph nodes and/or the spleenwhere they can educate other T-cells and form memory T-cells.

Example 20: Payload Delivery to DLL3+ Cancer Cells

CAR constructs are made using an anti-DLL3 antibody domain such asdescribed in US20170137533 (which is incorporated by reference in itsentirety for all purposes) as SC16.15. This anti-DLL3 antibody domain ismade into a single chain antibody (scFv), and the anti-DLL3 scFv iscombined with the transmembrane and intracellular portions of a CAR(such as those described in WO 2012/079000, which is herebyincorporated-by-reference in its entirety for all purposes) to make ananti-DLL3 CAR.

Payload constructs are made by engineering a transgene with an RDE sothat when the transgene is transcribed the transcript for the transgeneoperably links the transgene to the RDE. The payload transgene canencode an anti-4-1BB antibody, an anti-CD11b antibody, an anti-CTLA4antibody, an anti-IL1b antibody, a BiTE, a CCL2, an anti-CXCR4 antibody,an anti-CXCL12 antibody, a HAC, a heparinase, a hyaluronidase, a Hsp60,a Hsp70, an IL-2, an IL-12, an IL-15, an IL-18, an INFγ, a miRNA (e.g.,mir155), a CD40 ligand, an ApoE3, an ApoE4, an antagonists of CSF1receptor, a TNFα, and/or an anti-CD28 antibody. The RDE can be AU101(INFg) or AU14 (IL-6).

The constructs are transduced into primary T-cells which are thenallowed to return to a resting state. This produced anti-DLL3 CART-cells with one or more of the payloads: anti-CXCL12 antibody,anti-CXCR4 antibody, IL-12, anti-4-1BB antibody, anti-CD11b antibody,anti-CTLA4 antibody, anti-IL1b antibody, a BiTE, CCL2, HAC, heparinase,hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA (e.g.,mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies).

An NSG mouse model from Jackson Laboratories is used to establish cancerxenografts of human melanoma, human small cell lung cancer (SCLC), andhuman IDH1mut glioma. After the cancer xenograft is established in themice, the mice are treated with the primary T-cells with the anti-DLL3CAR and one of the payloads. Cancer xenograft killing is then comparedbetween the different payloads of the DLL3-CAR T-cells.

Example 21: B Toxin Fusion Payload Delivery to Target Cell

CAR T-cells are made as described in Example 19, except that the IL-12payload is replaced with a modified B Toxin fusion as described below.

The B Toxin fusion is made using tetanus toxin as described in Toivonenet al., Toxins 2:2622-44 (2010) and fusing the B domain of tetanus toxinto the payload encoding luciferase from Gaussia princeps (a secretedluciferase). This B Toxin payload is modified to provide for targetingto the CD19 antigen by replacing the tetanus B domain receptor bindingportion with the anti-CD19 CAR portion which binds to CD19 (e.g., ananti-CD19 scFv). This can be done as described in Kelley et al., Proc.Natl Acad. Sci. 85:3980-84 (1988) for the B domain of diphtheria toxin.

The primary T-cells with the anti-CD19 CAR and modified B Toxin payload(RDE controlled or constitutive) are administered to mice bearing CD19+tumors in their flanks. Killing of tumor cells and adverse responses aremonitored in the treated mice. The mice treated with the CAR T-cellswith RDE controlled, modified B Toxin payload show increased tumor cellkilling.

Example 22: hnRNPLL Control of Payload Expression

A nucleic acid encoding GFP is engineered to have extra introns and anextra exon at the 3′end of the coding region. The nucleic acids encodingGFP are followed by intron 1 from betaglobin, exon 4 of CD45 (hashnRNPLL binding sites) and another intron of betaglobin (engineered tohave hnRNPLL CA repeat binding sites). This engineered GFP encodingnucleic acid is operably linked to a constitutive promoter.

The expression cassette with the nucleic acid encoding this engineeredGFP is transduced into primary T-cells which are then allowed to expressthe GFP in a quiescent state. This should produce a GFP protein with theamino acids of exon 4 of CD45 on carboxyl terminal end. This fusionpolypeptide should have reduced GFP activity. The primary T-cells withthe expression cassette for GFP are then activated with anti-CD3/CD28beads. These activated cells should produce hnRNPLL which should lead toexclusion of exon 4 of CD45 from the GFP transcript which now encodesactive GFP. Translation of this transcript produces active GFP that canbe detected.

Example 23: Coordinated Delivery of CXCL9 and an Anti-PD1 Therapy toDLL3+ Cancer Cells

An anti-DLL3 CAR is made as described in Example 20. This CAR constructis engineered into T-cells also as described in Example 20.

Two payload cassettes are made for delivery by the anti-DLL3 CAR T-cell.First, a construct is made that encodes CXCL9 as a secreted payloadoperably linked to an RDE with an early expression profile (earlymaximal expression after activation of the cell) such as AU2 (CSF-2,maximal fold induction on day 1), AU101 (IFNg, maximal fold induction onday 1), or AU5 (EDN1, maximal fold induction on day 3/4). Second, aconstruct is made that encodes an anti-PD1 antibody (e.g., Pembrolizumab(Keytruda®)) as a secreted payload operably linked to an RDE with a lateexpression profile (late maximal expression after activation of thecell) such as AU22 (SEM-A4D, maximal fold induction on day 8) or AU19(TMEM-219, maximal fold induction on day 8). The two payloads can beplaced into a bicistronic construct, placed on the same construct, orthe payloads can be expressed from separate constructs. The payloadconstruct(s) are engineered into the anti-DLL3 CAR T-cell as describedabove in Example 20.

When this engineered CAR T-cell is administered to NSG mouse model asdescribed in Example 20. The CAR T-cells are activated by DLL3 at thetumor target, and the RDE constructs with the CXCL9 express this payloadfirst, and then at a later time the anti-PD1 antibody payload isexpressed. The AU2, AU5 or AU101 RDE of the CXCL9 construct has an earlymaximal expression of about 1 day after activation of the cell by DLL3at a cancer target. The CXCL9 can be secreted early after activation ofthe T-cell by DLL3 and the CXCL9 can potentiate the T-cell responses totumors treated with anti-PD1 antibodies. After CXCL9 secretion, anti-PD1is maximally secreted at a later time (about 8 days) and the effect ofthis antibody can be increased by the pretreatment with CXCL9.

The early expression of CXCL9 potentiates the activity and cancerkilling from the anti-PD1 antibody.

Example 24: Combination Therapy

CAR constructs are made using an anti-DLL3 antibody domain such asdescribed in US20170137533 (which is incorporated by reference in itsentirety for all purposes) as SC16.15 or SC16.25. This anti-DLL3antibody domain is made into a single chain antibody (scFv), and theanti-DLL3 scFv is combined with the transmembrane and intracellularportions of a CAR (such as those described in WO 2012/079000, which ishereby incorporated-by-reference in its entirety for all purposes) tomake an anti-DLL3 CAR.

Payload constructs are made by engineering a transgene with an RDE sothat when the transgene is transcribed the transcript for the transgeneoperably links the transgene to the RDE. The payload transgene canencode an anti-4-1BB antibody, an anti-CD11b antibody, an anti-CTLA4antibody, an anti-IL1b antibody, a BiTE, a CCL2, an anti-CXCR4 antibody,an anti-CXCL12 antibody, a HAC, a heparinase, a hyaluronidase, a Hsp60,a Hsp70, an IL-2, an IL-12, an IL-15, an IL-18, an INFγ, a miRNA (e.g.,mir155), a CD40 ligand, an ApoE3, an ApoE4, an antagonists of CSF1receptor, a TNFα, and/or an anti-CD28 antibody. The RDE can be AU101(INFg) or AU14 (IL-6).

The constructs are transduced into primary T-cells which are thenallowed to return to a resting state. This produced anti-DLL3 CART-cells with one or more of the payloads: anti-CXCL12 antibody,anti-CXCR4 antibody, IL-12, anti-4-1BB antibody, anti-CD11b antibody,anti-CTLA4 antibody, anti-IL1b antibody, a BiTE, CCL2, HAC, heparinase,hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA (e.g.,mir155), CD40 ligand, ApoE3, ApoE4, TNFα, CCR2, CCR4/CXCL12,CXCR3+CXCL9, CXCL9, ACLY, antagonists of CSF1 receptor, miRNA for Tox(e.g., hsa-mir-26b-5p (MIRT030248) hsa-mir-223-3p (MIRT054680)), miRNAfor TCF-7 (e.g., mIR-192, mIR-34a, miR-133a, miR-138-5p, miR-342-5p,miR-491-5p, miR-541-3p), and/or anti-CD28 antibody (including fulllength and fragments such as single chain antibodies).

An antibody drug conjugate (ADC) is made between an anti-DLL3 antibodysuch as described in US20170137533 (which is incorporated by referencein its entirety for all purposes) as SC16.15 or SC16.25. This anti-DLL3antibody domain is converted to an appropriate format (e.g., a Fab,F(ab′)2 or full-length IgG) and conjugated to one or more drugs (e.g.,etoposide, irinotecan, cisplatin and/or carboplatin).

An NSG mouse model from Jackson Laboratories is used to establish cancerxenografts of human melanoma, human small cell lung cancer (SCLC), andhuman IDH1mut glioma. After the cancer xenograft is established in themice, the mice are treated with the primary T-cells with the anti-DLL3CAR and one of the payloads, anti-DLL3 ADC, or primary T-cells with theanti-DLL3 CAR and one of the payloads and the anti-DLL3 ADC. Cancerxenograft killing is then compared between the ADC, different payloadsof the DLL3-CAR T-cells, and the different payloads of the DLL3 CART-cells with the anti-DLL3 ADC.

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for delivering a payload, comprising thesteps of: exposing a cancer cell to a therapeutic agent; obtaining aprimary T-cell comprising a chimeric antigen receptor, and aheterologous nucleic acid comprising a promoter operably linked to apolynucleotide encoding the payload that is operably linked to apolynucleotide encoding a RNA degradation element (RDE), wherein the RDEis an AU rich element, wherein the heterologous nucleic acid istranscribed to make a transcript encoding the transgene operably linkedto the RDE; exposing the primary T-cell to a ligand for the chimericantigen receptor wherein the ligand is associated with the cancer cell,and wherein binding of the ligand by the receptor activates the primaryT-cell; and expressing the transgene wherein the amount of polypeptidemade from the transgene is increased after the primary T-cell isactivated.
 2. The method of claim 1, wherein the therapeutic agent is anunconjugated antibody, an immunoconjugate, a gene therapy, achemotherapeutic agent, a therapeutic peptide, a cytokine, a localizedradiation therapy, a radioconjugate, a surgery, an interference RNA, adrug, or a toxin.
 3. The method of claim 2, wherein the therapeuticagent is an immunoconjugate and wherein the immunoconjugate binds to thesame ligand as the chimeric antigen receptor.
 4. The method of claim 2,wherein the therapeutic agent is an immunoconjugate and wherein theimmunoconjugate binds to a different ligand as the chimeric antigenreceptor.
 5. The method of claim 2, wherein the chemotherapeutic agentis a mitotic inhibitor, an antitumor antibiotic, a plant alkaloid, analkylating agent, an antimetabolite, and/or a radionuclide.
 6. Themethod of claim 1 wherein the transgene encodes a cytokine, a FasL, anantibody, a growth factor, a chemokine, an enzyme that cleaves apolypeptide or a polysaccharide, a granzyme, a perforin, or a checkpointinhibitor.
 7. The method of claim 1, wherein the transgene encodes anIL-2, and IL-12, an IL-15, an IL-18, an IFNg, a CD40L, or a TNF-α. 8.The method of claim 1, wherein the chimeric antigen receptor is ananti-DLL3 chimeric antigen receptor.
 9. The method of claim 8, whereinthe ligand is a DLL3 found on a tumor cell.
 10. The method of claim 9,wherein the tumor cell is an IDH1mut glioma cell, a melanoma cell, or asmall cell lung cancer cell.
 11. The method of claim 10, wherein thetransgene encodes a cytokine, a FasL, an antibody, a growth factor, achemokine, an enzyme that cleaves a polypeptide or a polysaccharide, agranzyme, a perforin, or a checkpoint inhibitor.
 12. The method of claim11, wherein the wherein therapeutic agent is an unconjugated antibody,an immunoconjugate, a gene therapy, a chemotherapeutic agent, atherapeutic peptide, a cytokine, a localized radiation therapy, asurgery, an interference RNA, a drug, or a toxin.
 13. The method ofclaim 11, wherein the therapeutic agent is an immunoconjugate andwherein the immunoconjugate binds to a DLL3.
 14. The method of claim 1,wherein the chimeric antigen receptor is an anti-TnMUC1 chimeric antigenreceptor.
 15. The method of claim 14, wherein the ligand is a TnMUC1found on a tumor cell.
 16. The method of claim 15, wherein the tumorcell is a breast cancer cell or a pancreatic cancer cell.
 17. The methodof claim 15, wherein the transgene encodes a cytokine, a FasL, anantibody, a growth factor, a chemokine, an enzyme that cleaves apolypeptide or a polysaccharide, a granzyme, a perforin, or a checkpointinhibitor, and wherein the wherein therapeutic agent is an unconjugatedantibody, an immunoconjugate, a gene therapy, a chemotherapeutic agent,a therapeutic peptide, a cytokine, a localized radiation therapy, asurgery, an interference RNA, a drug, or a toxin.
 18. The method ofclaim 1, wherein exposing the cancer cell to a therapeutic agent isperformed before exposing the primary T-cell to a ligand for thechimeric antigen receptor.
 19. The method of claim 1, wherein exposingthe primary T-cell to a ligand for the chimeric antigen receptor isperformed before exposing the cancer cell to a therapeutic agent. 20.The method of claim 1, wherein exposing the cancer cell to a therapeuticagent is performed at the same time as exposing the primary T-cell to aligand for the chimeric antigen receptor.